slowpoke: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - slowpoke
Cytological map position - 96A17
Function - Potassium channel
Symbol - slo
FlyBase ID: FBgn0003429
Genetic map position - 3-86
Classification - K[+]-channel-protein-Ca[2+]-activated
Cellular location - cell surface - transmembrane
|Recent literature||Li, X., Ghezzi, A., Krishnan, H. R., Pohl, J. B., Bohm, A. Y. and Atkinson, N. S. (2015). A histone modification identifies a DNA element controlling slo BK channel gene expression in muscle. J Neurogenet: 1-32. PubMed ID: 25967280
The slowpoke (slo) gene encodes BK type Ca2+-activated K+ channels. In Drosophila, expression of slo is induced by organic solvent sedation (benzyl alcohol and ethanol) and this increase in neural slo expression contributes to the production of functional behavioral tolerance (inducible resistance) to these drugs. Within the slo promoter region, it was observed that benzyl alcohol sedation produces a localized spike of histone acetylation over a 65 n conserved DNA element called 55b. Changes in histone acetylation are commonly the consequence of transcription factor activity and previously, a localized histone acetylation spike was used to successfully map a DNA element involved in benzyl alcohol-induced slo expression. To determine whether the 55b element was also involved in benzyl alcohol-induced neural expression of slo, it was deleted from the endogenous slo gene by homologous recombination. Flies lacking the 55b element were normal with respect to basal and benzyl alcohol-induced neural slo expression, the capacity to acquire and maintain functional tolerance, their threshold for electrically-induced seizures and most slo-related behaviors. Removal of the 55b element did however increase the level of basal expression from the muscle/tracheal cell-specific slo core promoter and produced a slight increase in overall locomotor activity. It is concluded that the 55b element is involved in control of slo expression from the muscle and tracheal-cell promoter but is not involved in the production of functional benzyl alcohol tolerance.
|Kadas, D., Ryglewski, S. and Duch, C. (2015). Transient BK outward current enhances motoneurone firing rates during Drosophila larval locomotion. J Physiol [Epub ahead of print]. PubMed ID: 26332699
A large number of voltage gated ion channels, their interactions with accessory subunits, and their posttranscriptional modifications generate an immense functional diversity of neurones. Therefore, a key challenge is to understand the genetic basis and precise function of specific ionic conductances for neuronal firing properties in the context of behavior. This study identifies slowpoke (slo) as exclusively mediating fast activating, fast inactivating BK current (ICF) in larval Drosophila crawling motoneurones. Combining in vivo patch clamp recordings during larval crawling with pharmacology and targeted genetic manipulations reveals that ICF acts specifically in motoneurones to sculpt their firing patterns in response to a given input from the central pattern generating (CPG) networks. First, ICF curtails motoneurone postsynaptic depolarizations during rhythmical CPG drive. Second, ICF is activated during the rising phase of the action potential and mediates a fast afterhyperpolarization. Consequently, ICF is required for maximal intraburst firing rates during locomotion, likely by allowing recovery from inactivation of fast sodium channels and decreased potassium channel activation. This contrasts the common view that outward conductances oppose excitability, but is in accord with reports on transient BK (see Drosophila Slowpoke) as well as Kv3 channel (see Drosophila Shawl) function in multiple types of vertebrate neurones. Therefore, this finding that ICF enhances firing rates specifically during bursting patterns relevant to behavior is likely of relevance to all brains.
|Krishnan, H. R., Li, X., Ghezzi, A. and Atkinson, N. S. (2016). A DNA element in the slo gene modulates ethanol tolerance. Alcohol 51: 37-42. PubMed ID: 26992698
In Drosophila, the slowpoke gene encodes BK-type Ca(2+)-activated K(+) channels and is involved in producing rapid functional tolerance to sedation with ethanol. Drosophila are ideal for the study of functional ethanol tolerance because the adult does not acquire metabolic ethanol tolerance. It has been shown that mutations in slo block the capacity to acquire tolerance, that sedation with ethanol vapor induces slo gene expression in the nervous system, and that transgenic induction of slo can phenocopy tolerance. This study used ethanol-induced histone acetylation to map a DNA regulatory element in the slo transcriptional control region. The chromatin immunoprecipitation assay was used to map histone acetylation changes following ethanol sedation to identify an ethanol-responsive DNA element. Ethanol sedation induced an increase in histone acetylation over a 60 n DNA element called 6b, which is situated between the two ethanol-responsive neural promoters of the slo gene. Removal of the 6b element from the endogenous slo gene affected the production of functional ethanol tolerance as assayed in an ethanol-vapor recovery from sedation assay. Removal of element 6b extended the period of functional ethanol tolerance from approximately 10 days to more than 21 days after a single ethanol-vapor sedation. This study demonstrates that mapping the position of ethanol-induced histone acetylation is an effective way to identify DNA regulatory elements that help to mediate the response of a gene to ethanol.
|Ding, Y., Berrocal, A., Morita, T., Longden, K. D. and Stern, D. L. (2016). Natural courtship song variation caused by an intronic retroelement in an ion channel gene. Nature 536: 329-332. PubMed ID: 27509856
Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Using an unbiased genetic approach, this study mapped a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, it was confirmed that slo is the causal locus, and the causal mutation was resolved to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.
| Saur, T., Peng, I.F., Jiang, P., Gong, N., Yao,
W.D., Xu, T.L. and Wu, C.F. (2016). K+
channel reorganization and homeostatic plasticity during postembryonic
development: biophysical and genetic analyses in acutely dissociated Drosophila
central neurons. J Neurogenet [Epub ahead of print]. PubMed
Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, this study shows drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons display prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) is far smaller in amplitude. In contrast, pupal neurons are characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removes inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents is accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolishes the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae is compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survive normally, Sh;;slo double mutants deficient in both fail to survive through pupal metamorphosis. Together, these data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.
|Kim, E. Z., Vienne, J., Rosbash, M. and Griffith, L. C. (2017). Non-reciprocal homeostatic compensation in Drosophila potassium channel mutants. J Neurophysiol: jn.00002.02017. PubMed ID: 28298298
Homeostatic control of intrinsic excitability is important for long-term regulation of neuronal activity. In conjunction with many other forms of plasticity, intrinsic homeostasis helps neurons maintain stable activity regimes in the face of external input variability and destabilizing genetic mutations. This study reports a mechanism by which Drosophila melanogaster larval motor neurons stabilize hyperactivity induced by the loss of the delayed rectifying K+ channel ShakerCognate B (Shab) , by upregulating the Ca2+-dependent K+ channel encoded by the slowpoke (slo) gene. Loss of SLO does not trigger a reciprocal compensatory upregulation of SHAB, implying that homeostatic signaling pathways utilize compensatory pathways unique to the channel that was mutated. SLO upregulation due to loss of SHAB involves nuclear Ca2+ signaling and dCREB, suggesting that the slowpoke homeostatic response is transcriptionally mediated. Examination of the changes in gene expression induced by these mutations suggests that there is not a generic transcriptional response to increased excitability in motor neurons, but that homeostatic compensations are influenced by the identity of the lost conductance.
|Bollinger, W. L., Sial, N. and Dawson-Scully, K. (2018). BK channels and a cGMP-dependent protein kinase (PKG) function through independent mechanisms to regulate the tolerance of synaptic transmission to acute oxidative stress at the Drosophila larval neuromuscular junction. J Neurogenet 32(3): 246-255. PubMed ID: 30484391
A cGMP-dependent protein kinase (PKG) has previously been shown to regulate synaptic transmission at the Drosophila neuromuscular junction (NMJ) during acute oxidative stress, potentially through modulation of downstream K(+) channel kinetics; however, the specific K(+) channels through which PKG functions remains unclear. It was hypothesized that PKG may be acting on calcium-activated large-conductance Slo K(+) channels, or BK channels. Genetic elimination and pharmacological inhibition of BK channel conductance increases synaptic transmission tolerance to acute H2O2-induced oxidative stress. Furthermore, it was discovered that activation of PKG in BK channel loss-of-function (Slo4) mutants significantly decreases time to stimulus-induced synaptic failure, providing the first evidence of PKG and BK channels functioning independently to control synaptic transmission tolerance to acute oxidative stress.
|Li, X., Abou Tayoun, A., Song, Z., Dau, A., Rien, D., Jaciuch, D., Dongre, S., Blanchard, F., Nikolaev, A., Zheng, L., Bollepalli, M. K., Chu, B., Hardie, R. C., Dolph, P. J. and Juusola, M. (2019). Ca(2+)-activated K(+) channels reduce network excitability, improving adaptability and energetics for transmitting and perceiving sensory information. J Neurosci. PubMed ID: 31350259
Ca(2+)-activated K(+) channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modelling, this study discover how visual information transfer in mutants lacking the BK channel (dSlo(-)), SK channel (dSK(-)) or both (dSK (-);;dSlo(-)) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K(+) conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. These dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca(2+)-activated K(+) channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.
|Gururaja Rao, S., Bednarczyk, P., Towheed, A., Shah, K., Karekar, P., Ponnalagu, D., Jensen, H. N., Addya, S., Reyes, B. A. S., Van Bockstaele, E. J., Szewczyk, A., Wallace, D. C. and Singh, H. (2019). BKCa (Slo) Channel Regulates Mitochondrial Function and Lifespan in Drosophila melanogaster. Cells 8(9). PubMed ID: 31438578
BKCa channels, originally discovered in Drosophila melanogaster as slowpoke (slo), are recognized for their roles in cellular and organ physiology. Pharmacological approaches implicated BKCa channels in cellular and organ protection possibly for their ability to modulate mitochondrial function. However, the direct role of BKCa channels in regulating mitochondrial structure and function is not deciphered. This study demonstrates that BKCa channels are present in fly mitochondria, and slo mutants show structural and functional defects in mitochondria. slo mutants display an increase in reactive oxygen species and the modulation of ROS affected their survival. The absence of BKCa channels reduced the lifespan of Drosophila, and overexpression of human BKCa channels in flies extends life span in males. This study establishes the presence of BKCa channels in mitochondria of Drosophila and ascertains its novel physiological role in regulating mitochondrial structural and functional integrity, and lifespan.
Slowpoke is a calcium-activated, voltage activated potassium channel (KCa channel) that functions to modulate ion flow in presynaptic nerve terminals, including those of the neuromuscular synapse. When a nerve fires, ions flow through channels in the presynapse, and this transfer of ions regulates release of neurotransmitter. Ion flow takes place in two phases: a falling phase, accompanied by Ca2+ entry into the presynaptic terminal (the inward current), and a rising phase, accompanied by K+ exit from the presynaptic terminal (outward current). Although it is difficult to determine whether this early Ca2+ current is itself sufficient to evoke transmitter release, it probably participates in the activation of Ca activated potassium channels, the subject of this essay. Inward currents are carried by voltage sensitive Ca channels, while outward currents are carried by Ca-activated, voltage activated potassium channels. Because KCa channel activity is regulated by both the membrane voltage and intracellular calcium, these channels can be thought of as a molecular locus for integrating electrical and biochemical signals (Yazejian, 1997 and references).
Voltage-gated potassium channels and the related calcium-activated potassium channels contain six putative transmembrane segments (S1-S6) in each subunit. The ß subunit of calcium-activated potassium channels (maxi K - Drosophila homolog Slowpoke) contains two putative transmembrane segments. Inwardly rectifying potassium channels appear to be distantly related to voltage-gated potassium channels and contain only two putative transmembrane segments (M1, M2) in each alpha subunit. The presence of intrinsic voltage sensors in voltage-gated potassium channels allows the activity of these channels to be controlled by membrane potential and enables them to control the waveform and firing patterns of action potentials. At least 18 genes for voltage-gated potassium channel subunits are known to be expressed in the mammalian nervous system. These genes belong to six subfamilies, corresponding to six Drosophila potassium channel genes (mammalian channels in parentheses): Shaker (Kv1.1-1.7); Shab (Kv2.1, 2.2); Shaw (Kv3.1-3.4); Shal (Kv4.1-4.3); Ether-a-go-go or Eag (HERG), and Slowpoke or slo (maxi K) (Jan, 1997 and references).
Large conductance calcium-dependent potassium channels, such as those belonging to the Slowpoke family, are ubiquitous in neurons and other excitable cells and play a critical role in regulating neuronal firing patterns and neurotransmitter release. The first KCa channel to be cloned was one encoded by the Slowpoke locus in Drosophila. The amino acid sequence of this Drosophila Slowpoke channel reveals that it is considerably longer than other voltage-dependent potassium channels, with an extended carboxy-terminal domain that constitutes about two-thirds of the channel protein. The function of this large domain is not clear, although evidence from mammalian Slowpoke homologs suggests that it may participate in calcium binding. Interestingly, the Drosophila and mammalian Slowpokes have a large number of alternative splice variants within this region of the protein (Zhou, 1999 and references).
Slob has been identified as a novel protein that binds to the carboxy-terminal domain of Slowpoke. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. 14-3-3 is known to be highly enriched in synaptic boutons at the neuromuscular junction and is present only at much lower levels in the motor axon and muscle (Broadie, 1997). Slob is also enriched in synaptic boutons, although its distribution appears to be less restricted than that of 14-3-3. Both 14-3-3 and dSlo are prominent in synaptic boutons, where they colocalize. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation of these residues. Slob itself increases the voltage sensitivity of dSlo, whereas 14-3-3 decreases the channel's voltage sensitivity (Zhou, 1999).
What are the molecular details of the profound downregulation of dSlo channel activity by 14-3-3? Members of the family of KCa channels are subject to modulation by a variety of molecular mechanisms, ranging from protein phosphorylation to oxidation/reduction reactions. It is conceivable that simply the binding of 14-3-3 to dSlo via Slob is sufficient to alter the gating of the channel, as appears to be the case for ß subunit interactions with Slowpoke and other potassium channels. Alternatively, 14-3-3 may act as another scaffolding component, to bring one of the protein kinases that it is known to bind into the proximity of the channel. Indeed, because 14-3-3 dimerizes, it might bridge the interactions of several different signaling proteins with the channel. It will be interesting to determine whether the Raf protein kinase, one of the kinases that binds 14-3-3, can phosphorylate and modulate dSlo, because Raf is a key player in the mitogen-activated protein (MAP) kinase pathway that conveys signals from the plasma membrane to the cell nucleus. Activation of this pathway can influence ion channel expression and activity, and potassium channel activity in turn can modulate tyrosine kinase signaling in cells. Thus, the present findings raise the intriguing possibility that a potassium channel regulatory complex is involved in MAP kinase signaling and the regulation of many fundamental cell processes (Zhou, 1999 and references).
The finding that the interaction of Slob with 14-3-3 requires Slob phosphorylation is consistent with studies of other 14-3-3 binding proteins. It is especially intriguing that the binding can be regulated in vivo by changes in the activity of CaMKII; these results suggest that there may be dynamic physiological regulation of dSlo channel activity by 14-3-3 that depends on the phosphorylation state of Slob. In view of the presynaptic colocalization of the three proteins described here, it is interesting that it is the CaMKII phosphorylation of Slob that regulates 14-3-3 binding. CaMKII is also present at a high concentration presynaptically in Drosophila, and thus the same calcium rise that evokes transmitter release might promote phosphorylation of Slob, binding of 14-3-3, and downregulation of dSlo (Zhou, 1999 and references).
14-3-3 eta is required for photoreceptor development in Drosophila, while another isoform, 14-3-3 epsilon, also influences photoreceptor development by regulating Ras-mediated signaling pathways. It is particularly interesting that flies lacking 14-3-3 zeta are severely impaired in an olfactory learning task (Skoulakis, 1996) and exhibit defects in basal synaptic transmission as well as in synaptic plasticity (Broadie, 1997). 14-3-3 zeta is enriched in presynaptic boutons of the neuromuscular junction (Broadie, 1997), consistent with a role in synaptic transmission. This presynaptic localization of 14-3-3 zeta is confirmed and in addition it colocalizes with both dSlo and Slob in presynaptic boutons. Slowpoke channels are also enriched in presynaptic endings in rat brain and frog neuromuscular junction, where they influence transmitter release. Since dSlo current contributes to membrane repolarization and helps to limit transmitter release from Drosophila nerve terminals (Gho, 1992), and 14-3-3 downregulates dSlo via Slob, it is plausible that there is greater nerve terminal dSlo current in 14-3-3 mutant flies and that this accounts for the decreased synaptic transmission seen in these mutants (Broadie, 1997). The possibility that a modulatory complex associated with a neuronal ion channel may influence synaptic transmission, and ultimately higher brain functions, is an attractive hypothesis for future investigation (Zhou, 1999 and references).
During courtship, males of Drosophila melanogaster and of many other species vibrate their wings, producing a 'lovesong'. This courtship song plays an important role in the mating ritual and has been implicated as a potential species recognition signal. cacophony (cac), a mutation affecting the courtship song in Drosophila, has been seen to cause temperature-sensitive (TS) abnormalities. cac codes for a voltage sensitive calcium channel. This essay will first discuss the phenotypic effects of cac mutation, then the effects of slowpoke mutation on the courtship song, and finally the role played by genetic variation in channels in Drosophila evolution.
When exposed to high temperatures (37 degrees), cac flies show frequent convulsions and pronounced locomotor defects. This TS phenotype seems consistent with the idea that cac is a mutation in a calcium-channel gene; it maps to the same X-chromosomal locus that encodes the polypeptide comprising the alpha-1 subunit of this membrane protein: Dmca1A. Previously, only one other voltage-sensitive calcium channel (Dmca1D) was known in Drosophila, but no behavioral defects have as yet been associated with variations at the autosomal locus encoding Dmca1D. Analysis of the courtship song of some other TS physiological mutants that are independent of cac shows that slowpoke mutations, which affect a calcium-activated potassium channel, cause severe song abnormalities. Certain additional TS mutants, in particular paralytic (parats1) and no-action-potential (napts1), exhibit subtler song defects. The results therefore suggest that genes involved in ion-channel function are a potential source of intraspecific genetic variation for song parameters, such as the number of cycles present in 'pulses' of tone or the rate at which pulses are produced by the male's wing vibrations during courtship. The implications of these findings from the perspective of interspecific lovesong variations in Drosophila are discussed. cacophony is one of the most interesting song mutations from an evolutionary point of view, at least in part because its abnormal pulses are nicely patterned, as in the case of wild-type males from various Drosophila species, and do not appear to be pathologically defective. A similar statement is possible about the songs of slowpoke males, although perhaps some of these mutant song bouts are more properly categorized as erratic and messy. Nevertheless, it is hard to believe that the song produced by double mutants cac;slo1/slo1 comes from D. melanogaster males, so striking are the differences from the wild-type patterns (Peixoto, 1998).
cacophony is a temperature-sensitive mutant: When exposed to high temperatures (~37°) cac flies show frequent convulsions and pronounced locomotor defects. This convulsion phenotype is characterized by flies turning upside-down or on their sides, shaking their legs for a few seconds, and then turning right-side up. The flies also curl their abdomen severely, either when on their backs or when walking, and twist their bodies at the same time. In addition, occasionally the cac adults will walk sideways, spin around on the same spot for a couple of seconds (apparently completely disoriented), leap across the chamber, or jump and tumble up and down out of control. There was no obvious sequence in the occurrence of these phenotypes. After long exposures at 37°, cac flies spend more and more time on their backs, shaking the legs until they seem to collapse. This typically requires more than 1 hr of heating for 1-day-old flies, but much less for older ones. As long as leg movement is still occurring, the mutant individuals usually recover in a few minutes after transfer to room temperature (Peixoto, 1998).
Only pulse song was examined in this report (courtship hums, or sine-song, being another type of song). Usually, all the pulses of the song of a given fly are logged, that is, marked for storage in the relevant file using the computer as an event-recorder, while scanning the visual record of the song along with the video image of the flies' behavior. Logging of some songs extended for only 2 min, and more than 500 pulses were typically logged. Songs with less than 40 pulses were not included in the analysis. Four parameters of the flies' pulse song were measured: interpulse interval (IPI), Cycles-per-Pulse (CPP), amplitude, and intrapulse frequency (IPF). CPP and IPF values can vary together among Drosophila types, but there is no way to predict one value from knowledge of the other; thus, these were treated as separate song parameters. The pulse amplitude measurements were attempts to quantify a song's loudness. This is difficult to measure reliably, and the units specified are arbitrary (Peixoto, 1998).
To examine the effects that temperature variation might have on the pulse song produced by cac, a song analysis of cac and wild-type flies was carried out at temperatures ranging from 15 to 30 degrees in steps of 2.5 degrees. Also included in this analysis was the mutant parats1, because a preliminary analysis had found it to have an effect on song at 25 degrees. Four pulse-song parameters were examined: amplitude of sound, IPI, CPP, and IPF. Temperature has a major effect on amplitude and IPI of all three genotypes, although it is far less clear in the case of CPP and IPF, even though the temperature effect is significant for the latter. Significant genotype differences were observed for amplitude, IPI, and CPP but not for IPF. The results also show the basic differences between cac mutation songs and wild-type (normal) songs, that is, higher amplitude and CPP, as well as longer IPIs in the former compared to the latter. IPFs are similar between these two genotypes. Although the overall trend observed for amplitude and IPI is similar for wild-type, cac, and para (as the temperature rises, there is an increase in the former and a decrease in the latter), differences were revealed in the way the various types of males react to temperature. These differences are responsible for the significant genotype x temperature interactions observed. The difference in IPI between cac and wild type shows a significant negative correlation with temperature. The difference is actually larger at lower temperatures, a result that is somewhat counterintuitive if one considers that the convulsion phenotype of this mutant occurs at elevated temperatures. It is possible that this reflects in part the nonlinear nature of the IPI change with temperature. No significant correlation with temperature was observed for the amplitude differences. The difference in IPI between parats1 and wild type shows the opposite trend observed for cac mutants. There is a significant positive correlation of temperature with the larger IPI difference at 30°. In the case of amplitude, however, the differences between parats1 and wild type show a significant negative correlation (Peixoto, 1998).
The mutant alleles, slo1 and slo2 define slowpoke as a new courtship-song gene. The sounds produced by these two mutants are clearly aberrant in the pulse songs produced, and they are in fact often difficult to log due to the low-amplitude or polycyclic nature of pulses (at a given moment of singing). Using the same criteria and IPI cutoffs used with the other mutants, all four song parameters examined are affected by these two slo alleles, which cause somewhat distinct song abnormalities. Males homozygous for the slo1 mutation produce very low-amplitude songs with long IPIs, and low CPP and IPF values. Isolated putative pulses, usually monocyclic signals, often occur in slo1 song records; however, they were not logged because they did not occur in pulse trains. In the case of the slo2 allele, the IPIs of homozygous mutant males are not as long, and the sound amplitude not as low, as in the case of slo1. A train of pulses in the song produced by flies homozygous for slo2 often ends with a highly polycyclic pulse. In fact, the mean number of cycles per pulse of slo2 flies is higher than the wild-type control. Isolated pulses were also often observed, but in this case (cf. slo1) they are usually highly polycyclic. Heterozygous flies slo1/slo2 show effects intermediate between the two homozygotes. The differences in the phenotypes between the two mutants obviously suggest differences in the molecular nature of the lesions that are unknown. slo1 is a chemically induced mutation, while slo2 was generated using gamma rays (Peixoto, 1998). Neither shows any gross chromosomal rearrangements (N. S. Atkinson, personal communication to Peixoto, 1998).
A fair fraction of the song mutants resulting from changes in genes that have been characterized at the molecular level involve membrane excitability. Not surprisingly, these basic functions, when mutated, lead to grossly appreciable defects in behavior. Only some of these mutants are song-defective as well. cacophony now finds itself in this category, that is, the courtship variant mutant that started out as a song mutant but is now known to have other phenotypic defects, such as heat-induced convulsions. This kind of general impairment could be at least as detrimental to fitness as the song abnormalities produced by cac mutants. Other pleiotropic song mutants with molecular correlates involve the regulation of gene expression (considered in general terms: transcription or RNA processing). In addition to the period and dissonance mutants in this category, consider the fruitless gene and its mutants. These courtship mutations defined a locus encoding a transcription factor. fru mutations affect courtship song, as well as other aspects of the fly's reproductive behavior, including fertility. Pleiotropies of these sorts place important constraints on the evolution of these behavioral genes (Peixoto, 1998 and references).
Genetic variation for features of the Drosophila courtship song have been reported from natural populations. It is possible that the level of genetic variability observed is influenced not only by sexual selection acting on the song parameters themselves, but also by selection on the pleiotropic effects of these putative song genes. These pleiotropic effects could even include other aspects of the mate recognition system. For example, there are smellblind mutations at the para locus (Lilly, 1994) that affect the response of males to female pheromones. It is also conceivable that directional selection acting on some of these pleiotropic effects, for example, selection for temperature tolerance and ion-channel genes, could drive changes in the song repertoire that could eventually lead to reproductive isolation between different populations (Peixoto, 1998 and references).
While the constraints associated with pleiotropy certainly do not prevent the rapid evolution of Drosophila courtship songs, it might explain why there is little evidence for genes with major effects on song found in crosses between closely related species. It is likely that the lovesong differences between most such species are based on the cumulative effect of very mild and subtle changes in several genes, at least a handful of them involving, for example, interspecific variations at the cac, slo, and mle (nap) loci. The major innovations in song production in the genus Drosophila seem to have occurred among Hawaiian flies for which founder-effect models of speciation have been proposed. These include, for example, the idea of fixation of a mutation in a major locus, via genetic drift, followed by selection for modifiers on its deleterious effects. Pleiotropy and epistasis have major roles in these models. Epistasis between conspecific genes is a key component of this sexually related phenotype. Epistatic interactions among song genes, such as the one found between cac and napts1 within D. melanogaster, could also have important implications for sexual selection on the phenotypes they control and on their potential role in speciation. Because of the role acoustic signals (such as the Drosophila's lovesong) play in female receptivity, mating preferences, and sexual isolation between species, song factors are among the best candidates for the so-called 'speciation genes'. The behavioral analysis presented here reveals that mutations in loci affecting ion-channel function might be a source of genetic variation in the fly's lovesong. Because of their enormous diversity, channel genes might turn out to be among the most common classes of song genes (Peixoto, 1998 and references).
Drosophila Slowpoke (Slo) calcium-dependent potassium channels bind directly to the catalytic subunit of cAMP-dependent protein kinase (PKAc). Coexpression of PKAc with Slo in mammalian cells results in a dramatic decrease of Slo channel activity. This modulation requires catalytically active PKAc but is not mediated by phosphorylation of S942, the only PKA consensus site in the Slo C-terminal domain. Slo binds to free PKAc but not to the PKA holoenzyme that includes regulatory subunits and is inactive. Activators of endogenous PKA that stimulate Slo phosphorylation, but do not produce detectable PKAc binding to Slo, do not modulate channel function. Furthermore, the catalytically inactive PKAc mutant does bind to dSlo but does not modulate channel activity. These results are consistent with the hypothesis that both binding of active PKAc to dSlo and phosphorylation of dSlo or some other protein are necessary for channel modulation (Zhou, 2002).
The initial evidence on modulation of calcium-dependent potassium channels by closely associated protein kinases or phosphatases came from experiments in which KCa channels reconstituted from rat brain were activated in an artificial lipid bilayer by the addition of ATP. PKAc was identified as one of several protein kinases closely associated with Slo channels. To determine whether the bound PKAc modulates channel function, Slo currents were recorded in the whole-cell voltage-clamp configuration. There is a dramatic downregulation of Slo channel functional activity with cotransfection of PKAc. Although it has been reported that channel function is not affected by coexpression with both PKAc and Src, it is now known that Src enhances Slo channel activity, and this upregulation by Src may have masked the PKAc downregulation that is described in this study (Zhou, 2002).
The reduction of whole-cell Slo in PKAc-cotransfected cells represents a change in channel functional activity. Although it is difficult to obtain an accurate conductance-voltage relation because of the large current amplitude in the whole-cell configuration, both the lower current amplitude and slower activation kinetics of Slo currents in PKAc-cotransfected cells are consistent with a decreased sensitivity of the Slo channel to voltage and calcium. The changes in current do not appear to result from alterations in channel expression or membrane targeting, because the expression level of Slo protein, measured by quantitative Western blot, is actually several-fold higher, and its surface localization measured by immunocytochemistry is unchanged, in PKAc-cotransfected cells. In any event, it is difficult to explain the change in channel kinetics in terms of protein expression level (Zhou, 2002).
Although questions still remain about the precise molecular mechanism underlying the profound modulation of Slo channel activity by cotransfected PKAc, the studies suggest strongly that it requires the association between active PKAc and the Slo channel. However, Slo channel activity is not altered by cotransfection of a catalytically inactive PKAc (K72E), although this mutant PKAc is capable of binding to Slo. This demonstrates that the modulation of Slo activity is not simply the result of binding of PKAc per se, but also requires phosphorylation. This is also consistent with other reports of modulation of native and expressed Slo family channels by PKAc-dependent phosphorylation. However, results with forskolin suggest that phosphorylation, although necessary, is not by itself sufficient to produce modulation. Accordingly, the hypothesis that modulation requires phosphorylation, of either Slo or some other protein, by active PKAc bound to the channel, is favored (Zhou, 2002).
To determine the potential molecular target the phosphorylation of which might mediate the modulation of Slo activity, the role of serine 942 in the Slo C-terminal domain was examined. This residue has long been recognized as a consensus PKA substrate, and it is readily phosphorylated by PKA both in vitro and in vivo. Interestingly, it was found that the mutation of serine 942 to alanine does not affect Slo binding to or modulation by PKAc. This is consistent with a previous report showing that S942A Slo is not different from the wild-type channel in its kinetic variability when expressed in Xenopus oocytes. Although it is convenient to use the anti-pS942 antibody to measure channel phosphorylation, this is not meant to imply that S942 participates in Slo modulation by PKAc (Zhou, 2002).
In addition to S942, other serine and threonine residues are thought to be exposed to the intracellular milieu. To identify other PKA substrate sites on Slo, an in vitro phosphorylation assay was performed using a recombinant fusion protein containing the entire Slo C-terminal domain. This domain is considerably longer than many other voltage-dependent potassium channels and constitutes approximately two-thirds of the total Slo channel protein. Somewhat surprisingly, it was found that other than S942, no additional amino acid in the entire Slo C-terminal domain is phosphorylated by PKAc in vitro. However, PKA phosphorylation of several serine or threonine residues in the intracellular loops between transmembrane domains has not been examined. It is equally plausible that PKAc phosphorylates other proteins that may themselves or through some signaling pathway modulate Slo channel activity. Slo is indeed regulated by several closely associated proteins. At least one Slo-interacting protein, 14-3-3, interacts with Slo via the adaptor protein Slob in a phosphorylation-dependent manner, and formation of this protein complex results in inhibition of channel activity. A recent report also showed that a Slo channel associating protein, cPLA2-alpha, is a target for phosphorylation. Phosphorylation of cPLA2-alpha results in activation of the channel, whereas phosphorylation of other regulatory elements causes channel inactivation. Finally, Slo channel activity is not modulated by activators of endogenous PKA, including forskolin and cBIMPS, which do not enhance binding of PKAc to Slo. Although these results do not lend themselves readily to unequivocal interpretation, they are consistent with the hypothesis that whatever the actual substrates of PKA are, targeting of sufficient PKAc via binding to Slo is also a requirement for channel modulation (Zhou, 2002).
It has been well documented that PKA forms regulatory complexes with some ion channels and ligand-gated receptors. PKA targeting is often achieved through AKAPs, proteins that bind to the PKA regulatory subunit as well as the substrate. However, overlay experiments showed a direct binding between Slo and PKAc, suggesting a novel channel-PKA protein complex. The present studies support this hypothesis and provide additional molecular details about this regulatory complex. Using co-immunoprecipitation approaches, it was shown that Slo binds only to free PKAc but not to the PKA holoenzyme, and that both PKA regulatory subunit and PKI inhibit the association between Slo and PKAc. A 35 amino acid region has been identified in the Slo C-terminal domain that is essential for PKAc binding. Interestingly, this region includes the consensus PKA substrate site, S942, although residues within the substrate site itself (RRXS) do not appear to be critical for binding to PKAc. This demonstrates that the Slo-PKAc association is not simply an enzyme-substrate complex. It is also consistent with previous studies on interactions between PKAc and regulatory subunits or PKI that show that residues separate from the pseudosubstrate site of the regulatory subunits or PKI are important in mediating the high-affinity binding to PKAc. Moreover, in vitro phosphorylation results suggest that binding of Slo to PKAc does not prevent the enzyme from phosphorylating its substrates. It remains to be determined how the activity of Slo-bound PKAc is regulated in cells (Zhou, 2002).
It is noteworthy that channels of the Slo family can physically associate with other protein kinases, including the Src tyrosine kinase and type I cGMP-dependent protein kinase. This suggests that modulation of channel activity may involve multiple regulatory mechanisms. Further biochemical and electrophysiological studies to dissect these regulatory pathways will undoubtedly provide important insights into the relationship between various signal transduction pathways and neuronal physiology (Zhou, 2002).
The electrical properties of a cell are produced by the complement of ion channels that it expresses. To understand how ion-channel gene expression is regulated, the tissue-specific regulation of the slowpoke Ca2+-activated K+ channel gene has been studied. This gene is expressed in the central and peripheral nervous system, in midgut and tracheal cells, and in the musculature of Drosophila melanogaster. The entire transcriptional control region has been cloned previously and shown to reproduce the tissue and developmental expression pattern of the endogenous gene. slo has at least four promoters distributed over approximately 4.5 kb of DNA. Promoter C1 and C1c display a TATA box-like sequence at the appropriate distance from the transcription start site. Promoters C1b and C2, however, are TATA-less promoters. C1, C1b, and C1c transcripts differ in their leader sequence but share a common translation start site. C2 transcripts incorporate a new translation start site that appends 17 amino acids to the N terminus of the encoded protein. Deletion analysis was used to identify sequences important for tissue-specific expression. A transgenic in vivo expression system in which all tissues and developmental stages can be assayed easily was used. Six nested deletions were transformed into Drosophila, and the expression pattern was determined using a lacZ reporter in both dissected tissues and sectioned animals. Different sequences have been identified required for expression in the CNS, midgut, tracheal cells, and muscle (Brenner, 1996a).
The range of electrical properties that a neuron or muscle cell can manifest is determined by which ion channel genes it expresses and in what amounts. The Drosophila slowpoke Ca2+-activated K+ channel gene has four distinct promoters. The role that a downstream intronic region, called the C2/C3 region, plays in modulating Promoter C1 and Promoter C2 activity is assessed. Promoter C1 and Promoter C2 appear to be responsible for all neuronal and muscle expression, respectively. Transgenic flies were used to determine the expression pattern from each promoter in the presence and absence of the C2/C3 region. Deletion of this region silences Promoter C1 in adult but not larval CNS and causes a substantial reduction in Promoter C2 activity in adult but not larval muscle. The C2/C3 region also activates Promoter C1 in the animal's eye. By placing the C2/C3 region adjacent to a basal HSP70 promoter it has been demonstrated that the region contains elements that can specifically activate a heterologous promoter in the eye and in adult but not larval muscle. These results demonstrate that the C2/C3 region has a important role in regulating slowpoke developmental expression in the CNS and musculature and in regulating eye expression (Brenner, 1996b).
The slowpoke gene of Drosophila encodes a Ca2+-activated K+ channel that is expressed in neurons, muscles, tracheal cells and the middle midgut. The entire transcriptional control region of slowpoke is contained in 11 kb of genomic DNA. Previous work has identified four different tissue-specific promoters (Promoters C1, C1b, C1c and C2) and sequences that regulate their activity. The regulation of neuronal and muscle expression during embryogenesis is described and contrasted with its regulation during larval and adult stages. Embryonic regulation is fundamentally different. The embryo uses Promoter C1 and a previously undescribed promoter, called Promoter Ce, to drive neuronal expression. The expression patterns of these promoters are distinct. Muscle expression arises from Promoter C2 as in other developmental stages. A downstream intronic region has been shown to contain control elements that modulate promoter activity differently in embryos, larvae and adults. Embryonic CNS expression is not dependent on the intron, however; its deletion has substantial effects on neuronal expression in larvae and adults. In embryonic muscle, removal of the intron eliminates muscle expression even though this deletion does not reduce larval muscle expression (Thomas, 1998).
Transcriptional regulation of the Drosophila slowpoke calcium-activated potassium channel gene is complex. To date, five transcriptional promoters have been identified that are responsible for slowpoke expression in neurons, midgut cells, tracheal cells, and muscle fibers. The slowpoke promoter called Promoter C2 is active in muscles and tracheal cells. To identify sequences that activate Promoter C2 in specific cell types, small deletions were introduced into the slowpoke transcriptional control region. Using transformed flies, it was asked how these deletions affected the in situ tissue-specific pattern of expression. Sequence comparisons between evolutionarily divergent species helped guide the placement of these deletions. A section of DNA important for expression in all cell types was subdivided and reintroduced into the mutated control region, a piece at a time, to identify which portion is required for promoter activity. 55-, 214- and 20-nucleotide sequences that control promoter activity have been identified. Different combinations of these elements activate the promoter in adult muscle, larval muscle, and tracheal cells (Chang, 2000).
Based on the expression pattern of mutated reporter constructs, muscles could be grouped into four categories. The data indicate that each group differentially regulates Promoter C2. These groups are (1) larval muscle, represented by the larval body wall muscles; (2) adult asynchronous muscle, represented by the DLM and DVM flight muscle; (3) adult synchronous muscle, represented by the pleurosternal, basalare, pterale, and leg muscles; and finally, (4) jump muscle, represented by a single member, the TT muscle (Chang, 2000).
Evolutionary conservation was used as a rational approach for identifying important transcriptional control elements. Easily identifiable conserved blocks exist between the Promoter C2 control regions of D. melanogaster and D. hydei. Additional deletions were used to further cull unimportant from important sequences. The first, called BR17, removes nucleotides -975 to -401, while the second, called EX, removes nucleotides -400 to -62. In conjunction with the previously described P7 deletion, this provides an uninterrupted set of deletions that approach Promoter C2 from the 5' end. BR17 removes weakly conserved sequence and therefore might be expected to have little effect on Promoter C2 activity. Indeed, the BR17 deletion does not alter the muscle or tracheal cell expression pattern. Deletion EX, however, which removes the strongly conserved 55 box, the 4E region, and the 20 box, has a larger effect. This loss silences Promoter C2 in both adult asynchronous muscle and larval muscle groups. Low level expression persisted in most members of the synchronous muscle group. This is the first indication that some muscles differentially regulate Promoter C2 activity. Each conserved region was inserted back into the EX deletion construct and tested for the capacity to reactivate Promoter C2. The P6 construct represents the intact control region. Whereas the 55 box and the 4E region strongly stimulate larval muscle expression, only the 4E region stimulates expression in adult muscle. Promoter C2 is clearly regulated differently in larval and adult muscle (Chang, 2000).
Removal of the intronic region (+416 to +1473) reduces or eliminates expression in most adult flight muscle, but does not affect expression in larvae. This region includes the intron between exon C2 and C3 (downstream of the Promoter C2 tss) and portions of each exon. Unfortunately, this deletion alters the 5'-untranslated region and splicing of the mRNA encoding the reporter and consequently may alter the translatability or stability of the mRNA. Therefore, the loss of expression might not result from impaired transcription but from a change in mRNA stability (Chang, 2000).
The Gal4BII and Gal4B2.1 transgenes address this caveat. The former contains the intronic region in question, while the latter is lacking it. In both, exon C2 is directly tagged with a Gal4 reporter gene. Exon C2 is the first exon expressed by Promoter C2 and is not found in transcripts expressed by any of the other slowpoke promoters. Because the intronic region is downstream of the Gal4 insertion and not part of the reporter gene mRNA, its removal cannot affect message stability. Interestingly, in the Gal4B constructs, removal of the intronic region eliminates expression in adult asynchronous muscles but does not reduce expression in larval muscle. This is a second illustration of the difference in the regulation of larval and adult muscle groups. Expression in larval muscle is independent of the intronic region, while adult DLM and DVM expression is absolutely dependent on this fragment of DNA (Chang, 2000).
Even within the adult, distinct muscle subtypes show different sequence requirements. Adult thoracic muscles may be categorized as asynchronous or synchronous. Asynchronous flight muscles are optimized for generating force and rapid, repetitive, beating contractions. Neural stimulation makes this muscle competent for contraction but does not trigger a contraction. The synchronous muscles have fewer contractile fibers, a more developed SR, and serve to control flight and move the legs. In this subtype, excitation is tightly coupled to contraction. Asynchronous and synchronous muscle regulate Promoter C2 differently. When the C2/C3 intronic region was deleted (Gal4B2.1 construct), a loss of expression in the asynchronous DLM and DVM has been observed. The deletion does not, however, prevent expression in the synchronous pleurosternal, basalare, pterale, and leg muscles. A second, less robust, example of this dichotomy between asynchronous and synchronous muscle is provided by the EX deletion. EX eliminates expression in the asynchronous DLM and DVM but does not completely eliminate expression in the synchronous pleurosternal, basalare, pterale, and leg muscles (Chang, 2000).
A Gal4BII reporter gene provides a final example of muscle subtype regulation. The insertion of Gal4 into exon C2 causes a specific loss of expression in the TT muscle. This is a synchronous muscle that the animal uses to jump during flight initiation. Expression in other muscle types appears unaffected. The conclusion that Promoter C2 is normally active in the TT is based on the expression pattern of seven different reporter gene constructs and is not in question. The insertion must be responsible. In Gal4BII the structure of the message itself has been altered, which might affect the stability or translatability of the mRNA and results in the specific loss of expression in the TT. However, the most parsimonious explanation is that the insertion, which is adjacent to two evolutionarily conserved mef2 motifs, prevents the binding of factors required for expression in the TT but not in the other muscle types (Chang, 2000).
The slowpoke transcriptional control region is complex, containing at least five tissue-specific promoters. This complexity is mirrored in the regulation of a single slowpoke promoter; Promoter C2. The simplest model consistent with the results is as follows. (1) In general, promoter activation in muscle involves E boxes located in the flanking 4E and intronic regions. These may coordinate the binding of a muscle-activating transcription factor belonging to the myoD basic-helix-loop-helix superfamily. Adult tergotrochanter and asynchronous muscle regions have an absolute dependence for both regions. In larval body wall muscle, however, the intronic region is not required and the requirement for the 4E region can be supplanted by the 55 box. (2) Tracheal cell expression is not absolutely dependent on either of the E box regions that stimulate muscle expression. However, expression in these cells also employs a redundant system requiring the presence of either the 55 or the 20 boxes. The cis-acting 20 box is proposed to bind a transcription factor that stimulates tracheal cell but not muscle expression. It is therefore more specific than the 55 box. (3) It is possible that the capacity of the 55 box to stimulate expression in two very different larval cell types indicates that it participates in developmental stage rather than in tissue-specific stimulation and that it will enhance expression in any larval cell that does not actively prevent activation. However, it is not uncommon for a single transcription factor binding site to be involved in tissue-specific stimulation of transcription in distinctly different cell types (Chang, 2000).
The Drosophila slowpoke gene encodes a BK-type calcium-activated potassium channel. Null mutations in slowpoke perturb the signaling properties of neurons and muscles and cause behavioral defects. The animals fly very poorly compared with wild-type strains and, after exposure to a bright but cool light or a heat pulse, exhibit a 'sticky-feet' phenotype. Expression of slowpoke arises from five transcriptional promoters that express the gene in neural, muscle, and epithelial tissues. A chromosomal deletion (ash218), affecting the adjacent gene, has been identified that removes the slowpoke neuronal promoters but not the muscle-tracheal cell promoter. This deletion complements the flight defect of slowpoke null mutants but not the sticky-feet phenotype. Electrophysiological assays confirm that the ash218 chromosome restores normal electrical properties to the flight muscle. This suggests that the flight defect arises from a lack of slowpoke expression in muscle, whereas the sticky-feet phenotype arises from a lack of expression in nervous tissue (Atkinson, 2000).
In Drosophila, a number of key processes such as emergence from the pupal case, locomotor activity, feeding, olfaction, and aspects of mating behavior are under circadian regulation. To identify clock-controlled output genes, an oligonucleotide-based high-density array was used that interrogates gene expression changes on a whole genome level. Genes regulating various physiological processes were found to be under circadian transcriptional regulation, ranging from protein stability and degradation, signal transduction, heme metabolism, detoxification, and immunity. By comparing rhythmically expressed genes in the fly head and body, it was found that the clock has adapted its output functions to the needs of each particular tissue, implying that tissue-specific regulation is superimposed on clock control of gene expression. Finally, taking full advantage of the fly as a model system, a cycling potassium channel protein has been identified as a key step in linking the transcriptional feedback loop to rhythmic locomotor behavior (Ceriani, 2002).
The availability of a more complete description of clock-controlled genes enabled the selection of several candidates for the control of locomotor behavior. One of these candidates was Slowpoke binding protein (Slob), which binds to the Ca2+-dependent voltage-gated potassium channel Slowpoke (Slo). A mutation in the gene coding for this channel causes behavioral defects and an altered mating song, also a hallmark of certain clock components. slowpoke participates in the repolarization of the action potential in flight muscles and in motoneurons. Slob has been shown to modulate Slo activity per se, and through the formation of a complex with the zeta isoform of 14-3-3 protein that acts downstream in several signaling pathways (Ceriani, 2002).
slob mRNA cycles robustly in fly heads in LD and DD. This pattern was lost in the y w;;Clkjrk mutant background. Although slo was not detected as cycling by COSOPT because of its low level of expression, it was noticed that slo appears to cycle in phase with slob in both LD and DD. The cycling of slo was investigated by RT-PCR analysis, and the protein was shown to cycle and peak at ZT20 by Western blot. The slo spatial expression pattern has been studied extensively; slo mRNA is widely expressed in the adult brain. Furthermore, Slo protein has been localized both to neuronal cell bodies as well as to the neuronal projections (Ceriani, 2002).
Prompted by the speculation that Slo might be involved in circadian control of activity, the locomotor activity was examined in two slo mutants, slo I and slo 4. Wild-type flies show increased locomotor activity near dawn and dusk and remain quiescent the rest of the day. These bursts of activity do not merely follow the next temporal transition, but instead anticipate it. pero and Clkjrk mutants, which have defects in core clock components, behave differently from wild-type under entrained conditions. Although pero flies still look mostly rhythmic in LD, Clkjrk is often not. This apparent rhythmicity in pero flies is caused by the so-called 'startle effect,' an immediate behavioral response to the light/dark transitions. Most of the slo 4 mutant flies display weak rhythms (defined as lacking a consolidated peak in the periodogram analysis) or no rhythms at all in LD. As expected, the lack of rhythmicity persists under free-running conditions. Surprisingly, this arrythmicity is comparable to, if not worse than, the one displayed by Clkjrk (Ceriani, 2002).
slo I mutants, in contrast, display a milder phenotype, with only 40%-55% of rhythmic flies in LD and DD, respectively, which is commensurate with a hypomorphic slo mutation (as opposed to a true null, as is the case for slo 4). Given the nature of the slo 4 mutation and the difference in the strength of the phenotype observed between slo I and slo 4 mutants, slo 4/slo I trans-heterozygotes were tested to rule out the possibility that other loci (also affected by the chromosomal inversion) could be contributing to the observed phenotype. The slo 4/slo I mutants show a somewhat intermediate phenotype (especially obvious in DD) between that of slo 4 and slo I. A small number of slo4 heterozygotes (slo 4/+) were tested; most were either strongly or weakly rhythmic. No arrhythmic flies were found. This argues against an effect exerted by the other putative loci (Ceriani, 2002).
To determine whether this mutation causes a general decrease in motility, which by itself could result in arrhythmicity, the total locomotor activity displayed by the different genotypes under LD and DD conditions was quantified. Although wild-type flies appear to be slightly more active under constant darkness, both slo mutants are impervious to the lighting regimen. More importantly, the overall levels of activity are not different from those of the wild-type flies. The actograms of wild-type, slo 4, and slo I mutant flies were superimposed because the average activity plots are known to reveal features not apparent when individual flies are inspected. This analysis revealed that the most striking difference is the impaired anticipation of the transitions in the slo 4 (null) mutant flies, indicating that the temporal gating that consolidates behavior around dawn and dusk is absent in flies lacking slo function (Ceriani, 2002).
Microarray experiments are extremely powerful in their scope and should be taken as a starting point to delve into the specifics of different aspects of physiology that appear to be under control of the clock. Several genes were identified potentially linked to behavior. Follow-up of one of them, slo, implicates it as a central regulator of locomotor activity, because a null mutation (slo 4) in this locus results in behavioral arrhymicity without a major change in total activity levels. Several scenarios could account for these observations. A mutation in slo could cause arrhythmicity if it directly affects the output pathway controlling behavior by affecting the excitability of the neurons that control it, although if such were the case, hyperkinetic or hypokinetic flies would be expected. Alternatively, the mutation could act at the level of the pacemaker neurons by reducing the synchronous firing between the lateral neurons, which would also cause the observed lack of behavioral rhythmicity. slowpoke could also be 'gating' fly locomotor activity that would be regulated by additional unidentified components. The observation that slo 4 mutants lack the consolidation of behavior around dawn and dusk clearly favors this hypothesis, although additional work will be required to rule out other plausible scenarios, such as its involvement in the light input pathway that conveys environmental information to the clock or the core oscillator itself (Ceriani, 2002).
The notion that a potassium channel is involved in the generation of rhythmic activity was proposed a number of years ago after the analysis of membrane conductance changes in isolated retinal neurons of the mollusk Bulla. This observation, together with the finding that potassium currents are under circadian regulation in the mouse and that expression cycles in Kcnma1, the slowpoke mouse ortholog (Panda, 2002) strongly suggests that this mechanism of control of rhythmic activity could play a role in more complex organisms as well (Ceriani, 2002).
While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties. To test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons the molecular genetics of Drosophila was used to manipulate the expression of Eve in identified motoneurons. Increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, a decrease was observed in both the fast K+ conductance (IKfast) and amplitude of quantal cholinergic synaptic input. Charybdotoxin was used to pharmacologically separate the individual components of IKfast to show that increased Eve specifically down regulates the Slowpoke (a BK Ca2+-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRα-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRα-96Aa. Taken together, these findings demonstrate that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. These data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties (Pym, 2006).
The roles of different K+ currents in regulating the generation and waveform of action potentials in Drosophila dorsal longitudinal flight muscles (DLMs) were examined in current-clamp experiments. In response to depolarizing current, DLMs displayed an initial transient rectification of the electronic potential lasting for up to hundreds of milliseconds. This delay in excitation is followed by oscillations or graded spikes that finally gave way to sharply rising spikes. Previous voltage-clamp studies of DLMs have revealed an inward Ca2+ current and at least three K+ currents: IA and IK, which are voltage-dependent, and IC, which is Ca2+ dependent. IA and IC are early inactivating currents, while IK is a slow, noninactivating current. In mature adults, selective elimination of IA either with Shaker (Sh) mutations or with 4-aminopyridine (4-AP), has no effect on spike duration or on the delay in excitation. In contrast, when IC is specifically eliminated with the slowpoke mutation, there is no delay before excitation, the amplitude of the spikes is significantly increased, and the spike duration is increased by 10-fold. Similar results are obtained by reducing IC in normal muscle by intracellular injections of EGTA or by use of low Ca2+ saline. Furthermore, DLM spikes evoked in slo by stimulation of the motorneuron is also broadened, suggesting that IC functions in a similar manner during normal flight as in current-clamped muscles. Elimination of IK along with IA and IC in saline containing tetraethylammonium or Ba2+ results in further prolongation of the DLM spike. In Ba2+ saline, there is an additional increase in spike amplitude as well. It is concluded that in mature adults, IC, rather than IA, plays the major role in repolarization of DLM spikes and in the delay before excitation (Elkins, 1988).
The larval muscle fibers of Drosophila show four outward K+ currents in addition to the inward Ca2+ current in voltage-clamp recordings. The Shaker (Sh) and the slowpoke (slo) mutations, respectively, eliminate the voltage-activated fast K+ current (IA) and the Ca2(+)-activated fast K+ current (ICF). Quinidine specifically blocks the voltage-activated delayed K+ current (IK) at micromolar concentrations. Sh, slo and quinidine have been used to remove specifically one or more K+ currents, so as to study physiological properties of these currents not previously characterized, and to examine their role in membrane excitability. A linear relationship is observed between the peak ICF and the peak ICa at different membrane potentials. ICF inactivates considerably during a 140 ms pulse to +20 mV. Recovery from inactivation is not complete for up to 2 s at the holding potential of -50 mV, which is much slower than the recovery of Ca2+ current from inactivation. In addition to IA and ICF, two delayed K+ currents are also observed in these fibers, the voltage-activated IK and the Ca2(+)-activated ICS. Near the end of a 500 ms depolarizing pulse, both IA and ICF are inactivated. Ca2(+)-free and 20 mmol l-1 Ca2+ saline were used to examine the tail currents of the remaining IK and ICS. The tail currents of ICS are slower than those of IK and reverse between -30 and -50 mV in different fibers. The dose-dependence of the blockade of IK by quinidine, which does not indicate a simple one-to-one binding mechanism, was studied. Current-clamp recordings from normal, Sh, slo and the double-mutant Sh;slo fibers suggest that ICF plays a stronger role than IA in repolarization of the larval muscle membrane. Elimination of ICF facilitates the occurrence of action potentials. Further elimination of IK prolongs the action potentials to several hundred milliseconds (Singh, 1990).
In Drosophila, two Ca2(+)-activated K+ currents, ICF and ICS, have previously been distinguished in conventional voltage clamp experiments. The slowpoke (slo) mutation eliminates ICF specifically. In patch clamp recordings a single-channel Ca2(+)-activated K+ current is readily distinguished from other channel activities in normal larval muscle membrane, whereas no such current is observed in slo muscles. This single-channel current thus correlates with the macroscopic ICF. No obvious differences in amplitude or properties are detected between normal (+/+) and heterozygous (slo/+) ICF channels in whole-cell voltage clamp recordings or single-channel patch clamp recordings. These results are consistent with the hypothesis that slo is a structural gene for the ICF channels only under certain conditions. The selective effect of the slo mutation may reflect a defect in a regulatory mechanism that is specific for the functioning of the ICF channel protein (Komatsu, 1990).
A culture system of 'giant' Drosophila neurons derived from cytokinesis-arrested embryonic neuroblasts was developed to overcome the technical difficulties usually encountered in studying small Drosophila neurons. Cytochalasin B-treated neuroblasts differentiate into giant multinucleated cells that displayed neuronal morphology and neuron-specific markers. These giant neurons express different excitability patterns and membrane channels similar to those reported in excitable tissues of Drosophila. Individual neurons exhibit distinct all-or-none or graded voltage responses upon current injection. Both current- and voltage-clamp recordings could be performed on the same neuron because of the large cell size, thus making it possible to elucidate the functional role of the individual types of channels. By using pharmacological agents and ion substitution, the following currents were identified in these giant neurons: inward Na+ and Ca2+ currents and outward voltage-activated (the A-type and delayed rectifier) and Ca2+-activated K+ currents. In addition, a tetrodotoxin (TTX)-sensitive, Na(+)-dependent outward K+ current and a persistent component of an inward Na+ current, were observed which have not been reported in Drosophila previously. This culture system can be used to analyze the mutational perturbations in ion channels and the resultant alterations in membrane excitability. Neurons from the mutant slowpoke, which is known to lack a component of the Ca2+-activated K+ currents in muscles, exhibit prolonged action potentials associated with defects in the Ca2+-activated K+ current. This abnormality appears to be more severe in the neurites than in the soma (Saito, 1991).
In Drosophila muscles and neuronal cell bodies at least four different potassium currents have been identified whose activity shapes the electrical properties of these cells. Potassium currents also control repolarization of presynaptic terminals and, therefore, exert a major effect on transmitter release and synaptic plasticity. However, because of the small size of presynaptic terminals in Drosophila, it has not been possible to analyze the potassium currents they express. As a first approach to characterizing the ionic currents present at presynaptic motor terminals of Drosophila larvae, synaptic currents were measured at the neuromuscular junction. From the alterations in evoked synaptic currents caused by various drugs and by mutations known to affect potassium currents in other tissues, it is suggested that the repolarizing mechanism in presynaptic terminals consists of at least four distinct currents. One is affected by aminopyridines or Sh mutations, a second component is affected by the slo mutation, a third is sensitive to quinidine and one or more additional components are blocked by tetraethylammonium. Depolarization depends on a presynaptic calcium current, which displays only slight voltage-dependent inactivation. Because the mechanism of repolarization exerts a major effect on synaptic activity, this analysis provides a framework for further genetic and molecular dissection of the basic processes involved in the regulation of transmitter release (Gho, 1992).
Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches show potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increases steeply both with depolarization and with increasing calcium concentration. The single-channel conductance is 126 pS with symmetrical potassium concentrations. The mean open time of the channels is clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels (Adelman, 1992).
The slowpoke locus of Drosophila encodes a family of alternatively spliced mRNAs which encode large conductance calcium-activated potassium channels. Variability resides in blocks of amino acids designated boxes A, C, E, G, and I. Oocytes were injected with cRNAs that had been chosen for direct functional comparison of single box differences. Single channel records from inside-out patches of oocyte membranes expressing A1 or A3 forms, E1 or E2 forms, and G2-G5 forms were analyzed and compared. The main functional difference between A1 and A3 was in unitary conductance, whereas the main difference in properties between E1 and E2 was in calcium sensitivity. Activation kinetics were different between G3 and G5, but not consistently in different A and E box backgrounds. The results indicate that alternative splicing of a common RNA precursor contributes to the functional diversity of the expressed channel. These findings suggest that the variable region of the Slowpoke channel subunit comprises modular, yet interactive functional domains which influence the essential features of unit conductance, calcium sensitivity, and gating (Lagrutta, 1994).
High conductance, Ca2+-activated (BK-type) K+ channels from mouse (mSlo) and Drosophila (dSlo) differ in their functional properties but share a conserved core resembling voltage-gated K+ channels and a tail appended to the core by a nonconserved linker. The channel subunit is physically divisible into these two conserved domains and the core determines such properties as channel open time, conductance, and, probably, voltage dependence, whereas the tail determines apparent Ca2+ sensitivity. Both domains are required for function. The different roles of the core and tail have been demonstrated by taking advantage of the functional differences between mSlo and dSlo. Heterologous pairing of cores and tails from mSlo and dSlo show that single-channel properties are always characteristic of the core species, but that apparent Ca2+ sensitivity is adjusted up or down depending on the species of the tail. Thus, the tail is implicated in the Ca2+-sensing role of BK channels (Wei, 1994).
Reconstitution of large conductance calcium-activated potassium (KCa) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, KCa channels derived from the Drosophila slowpoke gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, the reconstitution of cloned and expressed Slo KCa channels from Xenopus oocyte membranes into lipid bilayers is described. The reconstituted channels demonstrate functional properties characteristic of native KCa channels. They possess a mean unitary conductance of approximately 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 microM Ca2+, their half-activation potential is near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo KCa channels are insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (KD = 158 microM, at 0 mV) and internal Ba2+ (KD = 76 microM, at 40 mV). In addition, they are blocked by internally applied 'ball' inactivating peptide (KD = 480 microM, at 40 mV). These results demonstrate that cloned KCa channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions (Perez, 1994).
Unitary currents were recorded from inside-out membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by beta functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocks with an apparent Kd of 80 microM at 0 mV and senses 0.18 of the membrane electric field; the voltage dependence lies entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this is not voltage-dependent. Currents through Y308V channels are very insensitive to any block by TEA; the apparent Kd at 0 mV is 26 mM and the blockade senses 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels show unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these correspond to inhibitions of approximately 80%, 55%, 25% and 10% (Shen, 1994).
Ionic currents are regulated by many conditions including disease states, aging, learning and memory, and chronic drug treatment. This study describes a novel phenomenon of regulation of ionic currents by developmental temperature. Developmental temperature selectively regulates a voltage-activated potassium current in Drosophila. Raising Drosophila larvae at 28 degrees C instead of 18 degrees C increases one of the two voltage-activated K(+)-currents, the delayed sustained IK, in their muscles by up to 3.5-fold, with little effect on the early transient current, IA. Consistent with this increase in IK, the amplitude and the duration of the action potentials are reduced. The major increase in IK occurs between a rather abrupt interval from 25 degrees to 28 degrees C. The activation curve of the increased current is shifted towards hyperpolarizing potentials. There is no change in activation kinetics. This phenomenon has mechanistic implications for activity-dependent neuronal plasticity, expression of ion channels in cultured cells and heterologous systems, phototransduction, and behavior. The specificity of the regulation suggests a discrete mechanism geared to affect excitability such that it can respond to altered external stimuli such as temperature (Chopra, 1994).
Cloned large-conductance Ca2+-activated K+ channels from Drosophila (dslo) and human (hslo) were expressed in Xenopus oocytes. The effects of Ca2+ and voltage on these channels were compared by analysing both macroscopic currents and single-channel transitions. The activation kinetics of dslo Ca2+-activated K+ channels are strongly influenced by the intracellular Ca2+ concentration, but are only minimally affected by membrane voltage. Current activation kinetics increase more than 60-fold in response to Ca2+ concentration increases in the range 0.56-405 microM, but increase less than 2-fold by voltage changes from -60 to +80 mV. The activation kinetics of hslo channels are similarly influenced by increases in Ca2+ concentration; however, these kinetics are also increased 5- to 10-fold by voltage changes from -60 to +80 mV. The deactivation kinetics of both dslo and hslo channels are also more Ca2+ sensitive than voltage sensitive. Increasing concentrations of Ca2+ slow deactivation kinetics more than 40-fold, while changes in the membrane voltage cause less than 2-fold changes. Ca2+ increases the activation kinetics by altering first latency distributions. Increasing the Ca2+ concentration from 0.56 to 2.4 microM causes a 20-fold decrease in the mean time to first channel opening. Both Ca2+ and voltage have large effects on regulating the steady-state open probability of these ion channels. Plots relating open probability (Po) to membrane voltage show a voltage dependence of 16.5 mV per e-fold change in Po for dslo and 12.3 mV per e-fold change in Po for hslo. At any given voltage the Ca2+ sensitivity of dslo is lower than that for hslo. The Hill coefficient for Ca2+ activation is 1.9 +/- 0.15, indicating that the binding of at least two Ca2+ ions is required to maximally activate both dslo and hslo channels. The gating kinetics of both dslo and hslo channels can be well described by three open and five closed states. Changing the free Ca2+ concentration alters the time constants for the three longest closed states, without affecting any of the open states. Changing the membrane voltage alters the same three closed states, as well as the longest of the three open states. The two shortest occupancy open and closed time constants underlying these states are largely independent of voltage and Ca2+. To account for these data, it is proposed that Ca2+ binding to the closed channel is the slow rate-limiting step in the activation pathway and, conversely, that Ca2+ unbinding is the slow rate-limiting step in the deactivation pathway. Hence, Ca2+ appears to bind to the closed channel and allows it to undergo a number of slow conformational changes that bring the channel to a state from which it can quickly open upon depolarization. These data imply that while both Ca2+ and voltage can alter the steady-state open probability of these channels, only Ca2+ has large effects on altering non-steady-state parameters and thus is the intracellular signal that predominantly modulates the rate of channel activation and deactivation (DiChiara, 1995).
Cloned large conductance Ca2+-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminates a putative cyclic AMP-dependent protein kinase phosphorylation site exhibit large, slow fluctuations in open probability with time. These fluctuations, termed 'wanderlust kinetics', occur with a time course of tens of seconds to minutes and have kinetic properties inconsistent with simple gating models. Wanderlust kinetics are still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution is increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics do not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics can be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrate up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels (Silberberg, 1996).
Using patch recording, the modulation by ATP gamma S of the cloned Drosophila slopoke calcium-dependent potassium channel (dSlo) expressed in Xenopus oocytes was examined. There is a large variation in the gating kinetics, open probability, and conductance level of the channel in this expression system, which complicates the analysis of modulatory events. Addition of ATP gamma S to the intracellular face of the patch does not consistently alter the overall open probability of dSlo, but it does increase the frequency of appearance of an exceptionally long-lived closed state of the channel. This modulation is not blocked by an inhibitor of several serine/threonine protein kinases, nor by mutation of a serine residue that is a target for phosphorylation by protein kinase A. Thus, ATP gamma S may alter dSlo kinetic properties by some mechanism other than serine/threonine phosphorylation (Bowlby, 1996).
Ion channel gene expression can vary substantially among neurons of a given type, even though neuron-type-specific firing properties remain stable and reproducible. The mechanisms that modulate ion channel gene expression and stabilize neural firing properties are unknown. In Drosophila , this study demonstrates that loss of the Shal potassium channel induces the compensatory rebalancing of ion channel expression including, but not limited to, the enhanced expression and function of Shaker and slowpoke. Using genomic and network modeling approaches combined with genetic and electrophysiological assays, it was demonstrated that the transcription factor Kruppel is necessary for the homeostatic modulation of Shaker and slowpoke expression. Remarkably, Kruppel induction is specific to the loss of Shal, not being observed in five other potassium channel mutants that cause enhanced neuronal excitability. Thus, homeostatic signaling systems responsible for rebalancing ion channel expression can be selectively induced after the loss or impairment of a specific ion channel (Parish, 2014).
This study provides evidence that the cell fate regulator Kr is a critical player in the compensatory control of potassium channel gene expression. It is speculated that the induction of Kr drives a pattern of gene expression, first used to establish neuronal identity in the embryo and then, postembryonically, to rebalance ion channel expression in the face of persistent or acute perturbation of the Shal channel. Surprisingly, Kr is induced after the loss of Shal, but not other potassium channel gene mutations that have been shown to cause neural hyperexcitability. It is concluded that Shal function is specifically coupled to a homeostatic feedback system that includes the Kr-dependent transcriptional response. As such, these data imply the existence of discoverable 'rules' that define how individual neurons will respond to mutations in ion channel genes. Recent work underscores the possibility that the regulation of ion channel expression can be conserved from Drosophila to mammalian central neurons. In Drosophila, the translational regulator Pumilio was shown to be necessary and sufficient for the modulation of sodium channel transcription after persistent changes to synaptic transmission in the CNS. More recent data indicate that Pumilio-2 regulates NaV1.6 translation in rat visual cortical pyramidal neurons in a manner consistent with that observed in Drosophila. In mammalian neurons, Kr-like genes (KLF) respond to neuronal activity and are studied intensively in the context of axonal regeneration, but a role in ion channel expression or homeostatic rebalancing has yet to be defined (Parish, 2014).
Kr and its homologs are potent regulators of neuronal cell fate. KLF4 and KLF5, in particular, have been shown to both maintain and reprogram embryonic stem cell fate (Sur, 2009). This study has provided evidence that Kr protein levels diminish to nearly undetectable levels in the postembryonic CNS. Kr expression is then induced to achieve potassium channel regulation. It is tempting to speculate that the rebalancing of ion channel expression postembryonically is a reinduction of the embryonic mechanisms that initially specify neuronal active properties (Parish, 2014).
Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, has been identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat Kv1.3. It is possible that Slob serves to cluster dSlo and EAG together in a complex. Indeed, an interaction between EAG and Slowpoke is predicted by the finding that eag mutants in Drosophila exhibit altered calcium-dependent potassium currents at the larval neuromuscular junction (Schopperle, 1998).
Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. When Slob and dSlo are expressed together, there is a striking change in these subcellular distributions. Although some dSlo remains membrane associated, the two proteins colocalize in large doughnut-shaped structures that do not correspond obviously to known cellular organelles. The identity of these doughnut structures has not yet been determined, and it cannot be ruled out that they result from overexpression in a heterologous system. Time-lapse video microscopy shows that the structures are dynamic, moving within the cell and undergoing division into smaller structures. Similar structures have been described in cells coexpressing Shaker potassium channels and SAP97, a postsynaptic density protein. One intriguing possibility is that Slob is involved in channel protein processing, targeting, and/or degradation, and that the unidentified structures contain dSlo channels on their way to or from the plasma membrane. Slob may represent a new class of multi-functional channel-binding proteins (Schopperle, 1998).
To test the possibility that Slob might influence dSlo channel activity, patches were excised from cells expressing dSlo, and a preparation of purified glutathione-S-transferase-Slob (GST-Slob) fusion protein was applied to the cytoplasmic side of the patch. GST-Slob application strongly increases the steady-state open probability of dSlo channels. This effect is rapid in onset and reverses readily upon washing. GST-Slob also increases the peak dSlo current evoked by depolarizing voltage pulses. Because dSlo is a calcium-dependent channel, calcium was buffered carefully to insure that the activation by GST-Slob is not due to a change in calcium concentration. Furthermore, GST-Slob has no effect on the activity of hSlo, which is also calcium dependent. This lack of activation of hSlo is consistent with the finding that mSlo (which is virtually identical in amino acid sequence to hSlo) does not coimmunoprecipitate with Slob. These results support the conclusion that Slob can interact specifically with the dSlo channel to cause a change in its functional properties (Schopperle, 1998).
What is the in vivo function of Slob? Analysis of the protein sequence provides no obvious answers. The sequence of Slob has not been reported previously, but it does contain several well-characterized sequence motifs. For example, it contains a leucine zipper region that may allow it to form a complex with itself or other leucine zipper-containing proteins. Alternatively, Slob may be involved in signaling. The closest sequence matches to Slob are the signaling enzymes PKC and guanylate cyclase, but the matches are weak and the catalytic residues necessary for these enzymatic activities are not found in Slob. There is a PKC phosphorylation consensus site in the TRKQ cassette that can be removed by alternative splicing, suggesting possible regulation of Slob by phosphorylation. Another possibility is that Slob is an adaptor molecule. Finally, several proline-rich motifs that might bind SH3 domains including a Src kinase binding motif present in only the larger of the predicted amino-terminal splice variants, can be identified in the Slob sequence. It is possible that Slob is required as an adaptor to bring signaling proteins in close proximity to dSlo. Slob and Src have been found to coimmunoprecipitate from cotransfected cells. Taken together with the signaling protein binding domains within the Slob amino acid sequence, the effects on channel subcellular localization and gating properties suggest that Slob might link multiple channel properties to diverse upstream signals (Schopperle, 1998). COPY BELOW TO CAMKII
Slob is a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium (KCa) channel. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. Coimmunoprecipitation experiments from Drosophila heads and transfected cells confirm that 14-3-3 interacts with dSlo via Slob. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation. 14-3-3 coexpression dramatically alters dSlo channel properties when wild-type Slob is present but not when a double serine mutant Slob that is incapable of binding 14-3-3 is present. The results provide evidence for a dSlo/Slob/14-3-3 regulatory protein complex (Zhou, 1999).
The neuromuscular junction develops rapidly in Drosophila embryos (Broadie, 1993), and synaptic boutons and other morphological features of the synapse can be observed readily in mature larvae. 14-3-3 is known to be highly enriched in synaptic boutons at the neuromuscular junction and is present only at much lower levels in the motor axon and muscle (Broadie, 1997). Slob is also enriched in synaptic boutons, although its distribution appears to be less restricted than that of 14-3-3. Both 14-3-3 and dSlo are prominent in synaptic boutons, where they colocalize. A truncated Slob that lacks the amino-terminal 101 amino acids does not coimmunoprecipitate with 14-3-3. An examination of the amino acid sequence of Slob (reveals two motifs in this amino-terminal domain that resemble sequences in other 14-3-3-binding proteins. Although the downstream proline residue is lacking in the two Slob motifs, the participation of these motifs in 14-3-3 binding was examined by mutating the second serine in each motif (S54 and S79) to alanine. Both S54A Slob and S79A Slob bind less well to 14-3-3 than does wild-type Slob. When the two mutations are combined, no interaction of S54A/S79A Slob with 14-3-3 can be detected. The S54A/S79A Slob and wild-type Slob bind equally well to dSlo. Thus, the S54 and S79 residues in Slob are essential for its binding to 14-3-3 but not to dSlo (Zhou, 1999).
To test the hypothesis that the interaction of Slob and 14-3-3 might be regulated dynamically by phosphorylation in the fly, transgenic Drosophila that exhibit either higher or lower CaMKII activity than wild-type flies were used. The RQED1 fly line expresses a constitutively active rat CaMKII under the control of a heat shock promoter. In contrast, the ala2 fly line expresses a peptide inhibitor of CaMKII, also under heat shock control. Expression of constitutively active CaMKII increases the binding of Slob and 14-3-3 relative to the nonheat-shocked control. In contrast, expression of the peptide inhibitor of CaMKII decreases the binding compared with the nonheat-shocked control. These results demonstrate clearly that the interaction of Slob and 14-3-3 is not static but can be influenced rapidly by changes in CaMKII activity in the fly. Slowpoke channel activity was examined in detached membrane patches from transfected cultured cells to test the possibility that the channel might be modulated by 14-3-3. dSlo current evoked by a depolarizing voltage step to +30 mV is not affected by the coexpression of 14-3-3 and is somewhat larger when the channel is coexpressed with Slob. In contrast, much less dSlo current is evoked by the same depolarizing voltage step when all three proteins are expressed together, even though the GFP fluorescence confirms robust expression and membrane targeting of dSlo in these cells. The mean relative peak conductance (GRel) evoked by voltage steps to +30 mV was 0.64 ± 0.05 (mean ± SEM, n = 6) in patches from cells expressing dSlo alone, 0.70 ± 0.04 in patches from cells coexpressing dSlo and 14-3-3, 0.81 ± 0.03 in patches from cells expressing dSlo and Slob, but only 0.29 ± 0.09 in patches from cells expressing dSlo, Slob, and 14-3-3. Thus, the dSlo current evoked by depolarization to +30 mV is inhibited about 65% by 14-3-3. To determine whether 14-3-3 binding to Slob is required for this effect, dSlo and 14-3-3 were coexpressed with the S54A/S79A mutant Slob that can bind dSlo but does not bind to 14-3-3. dSlo current in patches from these cells is essentially identical to that found in patches from cells transfected with dSlo and Slob. Thus, the effect of 14-3-3 on dSlo current must be via Slob (Zhou, 1999).
To investigate the mechanism of this modulation, the voltage dependence of dSlo channel activity was examined. Slob itself increases the voltage sensitivity of dSlo, whereas 14-3-3 decreases the channel's voltage sensitivity. This effect of 14-3-3 must be via Slob, because the voltage sensitivity in the presence of 14-3-3 and the S54A/S79A mutant Slob is identical to that seen when wild-type Slob is transfected alone. The shift in the voltage required for half-maximal activation (V1/2), elicited by 14-3-3, is 61 mV at 30 µM free calcium and is even larger at lower free calcium concentrations. These results also confirm the indication from the GFP fluorescence that dSlo channels are present in the membrane in the triply transfected cells, and it is their functional properties that are modulated by Slob and 14-3-3 (Zhou, 1999).
Large-conductance calcium-activated potassium channels (BK channels) are activated by depolarized membrane potential and elevated levels of intracellular calcium. BK channel activity underlies the fast afterhyperpolarization that follows an action potential and attenuates neurotransmitter and hormone secretion. Using a modified two-hybrid approach, the interaction trap, a novel protein from Drosophila, dSLIP1 (dSLo interacting protein), was identified which specifically interacts with Drosophila and human BK channels and has partial homology to the PDZ domain of alpha1 syntrophin. The dSLIP1 and dSlo mRNAs are expressed coincidently throughout the Drosophila nervous system, the two proteins interact in vitro, and they may be coimmunoprecipitated from transfected cells. Coexpression of dSLIP1 with dSlo or hSlo BK channels in Xenopus oocytes results in reduced currents as compared with expression of BK channels alone; current amplitudes may be rescued by coexpression with the channel domain that interacts with dSLIP1. Single-channel recordings and immunostaining of transfected tissue culture cells suggest that dSLIP1 selectively reduces Slo BK currents by reducing the number of BK channels in the plasma membrane (Xia, 1998b).
Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. Protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch (Esguerra, 1994).
Slob (Slowpoke-binding protein) modulates the Drosophila Slowpoke calcium-activated potassium channel. Slob deletion or RNAi knockdown has been shown to decrease excitability of neurosecretory pars intercerebralis (PI) neurons in the adult Drosophila brain. In contrast, this study found that Slob deletion/knockdown enhances neurotransmitter release from motor neurons at the fly larval neuromuscular junction, suggesting an increase in excitability. Because two prominent Slob isoforms, Slob57 and Slob71, modulate Slowpoke channels in opposite directions in vitro, this study investigated whether divergent expression patterns of these two isoforms might underlie the differential modulation of excitability in PI and motor neurons. By performing detailed in vitro and in vivo analysis, strikingly different modes of regulatory control by the slob57 and slob71 promoters was found. The slob71, but not slob57, promoter contains binding sites for the Hunchback and Mirror transcriptional repressors. Furthermore, several core promoter elements that are absent in the slob57 promoter coordinately drive robust expression of a luciferase vector by the slob71 promoter in vitro. In addition, the expression patterns of the slob57 and slob71 promoters was visualized in vivo, and clear spatiotemporal differences were found in promoter activity. Slob57 is expressed prominently in adult PI neurons, whereas larval motor neurons exclusively express Slov71. In contrast, at the larval neuromuscular junction, Slob57 expression appears to be restricted mainly to a subset of glial cells. These results illustrate how the use of alternative transcriptional start sites within an ion channel modulator locus coupled with functionally relevant alternative splicing can be used to fine-tune neuronal excitability in a cell-specific manner (Jepson, 2013).
Synaptic scaffold proteins control the localization of ion channels and receptors, and facilitate molecular associations between signaling components that modulate synaptic transmission and plasticity. This study defines novel roles for a recently described scaffold protein, Dsychronic (DYSC), at the Drosophila larval neuromuscular junction. DYSC is the Drosophila homolog of whirlin/DFNB31, a PDZ domain protein linked to Usher syndrome, the most common form of human deaf-blindness. DYSC is expressed presynaptically and is often localized adjacent to the active zone, the site of neurotransmitter release. Loss of DYSC results in marked alterations in synaptic morphology and cytoskeletal organization. Moreover, active zones are frequently enlarged and misshapen in dysc mutants. Electrophysiological analyses further demonstrate that dysc mutants exhibit substantial increases in both evoked and spontaneous synaptic transmission. Previous study have shown that DYSC binds to and regulates the expression of the Slowpoke (SLO) BK potassium channel. Consistent with this, slo mutant larvae exhibit similar alterations in synapse morphology, active zone size and neurotransmission, and simultaneous loss of dysc and slo does not enhance these phenotypes, suggesting that dysc and slo act in a common genetic pathway to modulate synaptic development and output. These data expand understanding of the neuronal functions of DYSC and uncover non-canonical roles for the SLO potassium channel at Drosophila synapses (Jepson, 2014).
slowpoke expression throughout development has been examined. It is expressed in muscle cells, neurons of the CNS and PNS, mushroom bodies, a limited number of cells in embryonic and larval midgut and in epithelial-derived tracheal cells. The promoter has been cloned and shown to direct expression in the same pattern as the endogenous gene in both neural and epithelial-derived cells. During pupariation and embryogenesis, slo is expressed in muscles many hours prior to the appearance of functional channels (Becker, 1995).
The entire developmental history of muscle membrane electrogenesis can be observed in the embryonic myotubes of Drosophila. The development of ionic currents and muscle properties was examined using whole-cell patch-clamp techniques throughout embryonic myogenesis. In the early stages of myogenesis, from myoblast fusion through to establishing epidermal insertions, the myotubes are electrically inert and are electrically and dye coupled to adjacent myotubes. Membrane electrogenesis begins in the mid-embryonic stages (early stage 16), when the myotubes abruptly uncouple, revealing the first of five prominent extrajunctional currents: a small, inward, voltage-gated calcium current (ICa). The uncoupling of the embryonic myotubes heralds the onset of extremely rapid electrogenesis; within several minutes both the fast, inactivating (IA; Shaker) and delayed, noninactivating (IK) outward potassium currents, the stretch-activated outward potassium current, and the junctional glutamate-gated inward current all appear and begin to develop in a current-specific manner. Very late in embryogenesis (late stage 17), the calcium-dependent, outward potassium currents [rapid, inactivating (ICF; slowpoke) then delayed, noninactivating (ICS)] develop, completing the complement of macroscopic currents in the mature larval muscle. Hence, the voltage-gated currents (ICa, IA, and IK, respectively) appear relatively early, and the calcium-dependent currents (ICF, ICS) appear only very late during myogenesis. This developmental progression of current maturation is reflected in dynamic changes in the voltage responses of the embryonic membrane, from wholly passive response to current injection in the early, coupled myotubes to regenerating, overshooting action potentials in the mature embryonic muscle. The earliest embryonic IA current has a midpoint of inactivation 40 mV more negative than the IA current in the mature embryo. As myogenesis proceeds, the inactivation curve develops a biphasic character, suggesting that a low-inactivation IA channel is present in early development and progressively replaced by the mature form as development proceeds. The current at all stages can be completely eliminated in Shaker mutants (ShKS133). These findings suggest that an embryonic form of the Shaker IA channel is present during early myogenesis. The prominent IA current present in early development is almost entirely inactivated at the physiological resting potential; the significance and mechanism of this developmental shift are unclear (Broadie, 1993).
The slowpoke gene of Drosophila encodes a pore-forming subunit of a BK-type CaCa2+-activated K+ channel. The gene is expressed in neurons, muscles, tracheal cells and in the midgut. The P1 transgene gene contains the entire slowpoke transcriptional control region and drives the expression of a reporter protein comprised of slowpoke amino terminal sequences fused to beta-galactosidase. Midgut expression is limited to the copper cell and iron cell regions. The copper cell region is composed of two cell types: the copper cells and the interstitial cells. The P1 transgene is expressed in the interstitial cells but not the copper cells. Furthermore, it is shown that the reporter protein is apically localized in the interstitial cells. In these cells, the slowpoke CaCa2+-activated K+ channel is thought to participate in the transport of ions between the hemolymph and the lumen of the gut. Subcellularly localized BK channels may be involved in the secretion of acid into the gut lumen. An analogous role for basolaterally localized BK channels has been proposed in the acid-secreting intercalating cells of the human kidney (Brenner, 1997).
Potassium channels have been implicated in central roles in activity-dependent neural plasticity. The giant fiber escape pathway of Drosophila has been established as a model for analyzing habituation and its modification by memory mutations in an identified circuit. Several genes in Drosophila encoding K+ channel subunits have been characterized, permitting examination of the contributions of specific channel subunits to simple conditioning in an identified circuit that is amenable to genetic analysis. Mutations altering each of four K+ channel subunits (Sh, slo, eag, and Hyperkinetic[HK]) have distinct effects on habituation at least as strong as those of dunce and rutabaga, memory mutants with defective cAMP metabolism. Habituation, spontaneous recovery, and dishabituation of the electrically stimulated long-latency giant fiber pathway response have been shown in each mutant type. Mutations of Sh (voltage-gated) and slo (Ca2+-gated) subunits enhance and slow habituation, respectively. However, mutations of eag and Hk subunits, which confer K+-current modulation, have even more extreme phenotypes, again enhancing and slowing habituation, respectively. In double mutants, Sh mutations moderated the strong phenotypes of eag and Hk, suggesting that their modulatory functions are best expressed in the presence of intact Sh subunits. Nonactivity-dependent responses (refractory period and latency) at two stages of the circuit are altered only in some mutants and do not account for modifications of habituation. Furthermore, failures of the long-latency response during habituation, which normally occur in labile connections in the brain, can be induced in the thoracic circuit stage in Hk mutants. This work indicates that different K+ channel subunits play distinct roles in activity-dependent neural plasticity and thus can be incorporated along with second messenger "memory" loci to enrich the genetic analysis of learning and memory (Engel, 1998).
Potassium channels control the repolarization of nerve terminals and thus play important roles in the control of synaptic transmission. The effects of mutations in the slowpoke gene are described on transmitter release at the neuromuscular junction in Drosophila. The slowpoke mutant exhibits reduced transmitter release compared to normal. Similarly, the slowpoke mutation significantly suppresses the increased transmitter release conferred either by a mutation in Shaker or by application of 4-aminopyridine, which blocks the Shaker-encoded potassium channel at the Drosophila nerve terminal. Furthermore, the slowpoke mutation suppresses the striking increase in transmitter release that occurs following application of 4-aminopyridine to the ether a go-go mutant. This suppression is most likely the result of a reduction of Ca2+ influx into the nerve terminal in the slowpoke mutant. It is hypothesized that the effects of the slowpoke mutation are indirect, perhaps resulting from increased Ca2+ channel inactivation, decreased Na+ or Ca2+ channel localization or gene expression, or by increases in the expression or activity of potassium channels distinct from slowpoke (Warbington, 1996).
Drosophila provides an excellent model for delineating the role of ion channels in the origin and transmission of heartbeat. Tests in Drosophila are reported on a wide range of mutations and pharmacological agents known to interfere with K+, Ca2+, Na+, and Cl- ion channels in well-characterized ways. K+ channels are central to heart function. Tetraethylammonium, which blocks all four K+ currents, slows the heart. Distinctions can be made among these currents. The mutation slowpoke and the agent charybdotoxin, both of which affect a fast CaCa2+-gated K+ channel, virtually eliminate heartbeat. Shaker and ether-a-go-go, which encode subunits of K+ channels, have moderate, possibly regulatory effects. 'OPQ-type' Ca2+ channels are critical. omega-Conotoxin MVIIC, which blocks these channels, virtually stops the heart. Amiloride, which may affect T-type Ca2+ channels, has no effect, nor do the L-type Ca2+ blockers verapamil and diltiazem. Temperature induced paralysis E, involved in the function of Na+ channels, the Na+ channel blockers tetrodotoxin and amiloride, and the Cl- blockers mefanamic and niflumic acids have no effect. Na+ and Cl- channels thus appear unnecessary for cardiac function (Johnson, 1998).
A mutation of Drosophila, slowpoke, specifically abolishes a Ca2+-dependent K+ current, IC, from dorsal longitudinal flight muscles of adult flies. Other K+ currents remain normal, providing evidence that IC is mediated by a molecularly distinguishable set of channels. The pharmacological properties of IC are similar to those of Ca2+-dependent currents in some vertebrate cells. The muscle action potential is significantly lengthened in slo flies, indicating that IC plays the major role in its repolarization (Elkins, 1986).
Plants produce insect repellents, such as citronellal, which is the main component of citronellal oil. However, the molecular pathways through which insects sense botanical repellents are unknown. This study shows that Drosophila uses two pathways for direct avoidance of citronellal. The olfactory coreceptor OR83b contributes to citronellal repulsion and is essential for citronellal-evoked action potentials. Mutations affecting the Ca2+-permeable cation channel TRPA1 result in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relies on a G protein (Gq)/phospholipase C (PLC) signaling cascade rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq, or PLC causes an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca2+-activated K+ channel (BK channel) Slowpoke results in a similar impairment in citronellal avoidance and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 is directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria (Kwon, 2010).
Two features of the citronellal responses were found to be abnormal in the trpA11 basiconic sensilla ab11a neurons. First, there was a higher citronellal-evoked action potential frequency than in wild-type. Second, there was a defect in deactivation in trpA11 ab11a neurons. The same two defective phenotypes were observed in ab11 neurons in the dGqα1 and norpAP24 mutants, although only the increase in the evoked responses was clearly different when testing significance by analysis of variance. These results support the conclusion that the dGqα1, norpAP24, and trpA11 mutations affect the citronellal response in an ORN in ab11 (Kwon, 2010).
The finding that there were increases in the frequency of citronellal-evoked action potentials was unexpected and raised a question as to the basis for these defects. TRPA1 is a Ca2+-permeable channel, and because reduced activity of Ca2+-activated K+ channels (BK channels) increases the frequency of action potential firing, it was of interest to see whether loss of TRPA1 caused reduced BK channel activity. If so, then a mutation in the gene (slowpoke, slo) encoding the fly BK channel might phenocopy the trpA1 phenotype. In support of this model, the slof05915 mutation caused an increase in the frequency of citronellal-evoked action potentials and impaired citronellal avoidance. Introduction of UAS-slo-RNAi in combination with either the trpA1-GAL4 or the Or83b-GAL4 resulted in a similar defect in citronellal avoidance (Kwon, 2010).
It is proposed that TRPA1 is required for activity of Slo, which in turn modulates citronellal-induced firing of action potentials. Slo might be required in many ORNs and be regulated by additional TRP channels. In support of this proposal, ab12 also responded to citronellal and displayed a higher frequency of action potentials in slof05915 but did not function through a Gqα/PLC/TRPA1 pathway. Knockout of a mammalian TRP channel, TRPC1, also disrupts the activity of a Ca2+-activated K+ channel (KCa) in salivary gland cells, and mutations affecting either TRPC1 or KCa result in similar defects in salivary gland secretion. Thus, a role for TRP channels in activating Ca2+-activated K+ channels might be a common but poorly appreciated general phenomenon that is evolutionarily conserved (Kwon, 2010).
The finding that loss of TRPA1 causes an increase rather than a decrease in citronellal-induced action potentials suggests that there might be a TRPA1 independent-pathway required for generating action potentials in response to citronellal. OR83b is a candidate for functioning in such a pathway, because mutation of Or83b interferes with the ability of the synthetic repellent DEET to inhibit the attraction to food odors. This study found that Or83b1 mutant flies, or Or83b1 in trans with a deficiency that uncovers the locus, exhibited an impairment in citronellal avoidance similar to that in trpA1 mutant flies. An Or83b1 defect in the DART assay was not specific to citronellal, because these flies were also impaired in the response to benzaldehyde. Tested were performed to see whether the frequency of citronellal-induced action potentials was altered in Or83b1 ab11 sensilla. In contrast to the trpA1 mutant phenotype, none of the mutant Or83b1 ab11 neurons responded to citronellal (Kwon, 2010).
These data indicate that there are dual pathways required for the response to citronellal. OR83b is necessary for producing citronellal-induced action potentials, and a Gq/PLC/TRPA1 pathway appears to function in the modulation of action potential frequency by activating BK channels. It is suggested that an abnormally high frequency of action potentials may lead to rapid depletion of the readily releasable pools of neurotransmitter, thereby muting the citronellal response. Interestingly, a loss-of-function mutation affecting a worm BK channel also results in a behavioral phenotype—increased resistance to ethanol. Although Drosophila TRPA1 functions downstream of a Gq/PLC signaling pathway, citronellal can also directly activate TRPA1, but with low potency. Nevertheless, because Anopheles gambiae TRPA1 is also expressed in the antenna and is activated directly by citronellal with high potency, it is suggested that mosquito TRPA1 represents a new potential target for in vitro screens for volatile activators that might serve as new types of insect repellents (Kwon, 2010).
Tethered flies allow studies of biomechanics and electrophysiology of flight control. Microelectrode recordings were performed of spikes in an indirect flight muscle (the dorsal longitudinal muscle, DLMa) coupled with acoustic analysis of wing beat frequency (WBF) via microphone signals. Simultaneous electrophysiological recording of direct and indirect flight muscles has been technically challenging; however, the WBF is thought to reflect in a one-to-one relationship with spiking in a subset of direct flight muscles, including muscle m1b. Therefore, this approach enables systematic mutational analysis for changes in temporal features of electrical activity of motor neurons innervating subsets of direct and indirect flight muscles. This paper reports the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at approximately 5 Hz) and the corresponding wing beat frequency (approximately 200 Hz). Mutants were examined of: 1) voltage-gated Ca2+ channels (cacophony, cac), 2) Ca2+-activated K+ channels (slowpoke, slo), and 3) voltage-gated K+ channels (Shaker, Sh) and their auxiliary subunits (Hyperkinetic, Hk and quiver, qvr). Flight initiation in response to an air puff was severely disrupted in both cac and slo mutants. However, once initiated, slo flight was largely unaltered, whereas cac displayed disrupted DLM firing rates and WBF. Sh, Hk, and qvr mutants were able to maintain normal DLM firing rates, despite increased WBF. Notably, defects in the auxiliary subunits Hk and qvr could lead to distinct consequences, i.e. disrupted DLM firing rhythmicity, not observed in Sh. This mutant analysis of direct and indirect flight muscle activities indicates that the two motor activity patterns may be independently modified by specific ion channel mutations, and that this approach can be extended to other dipteran species and additional motor programs, such as electroconvulsive stimulation-induced seizures (Iyengar, 2014).
Six mutants of SLO-1, a large-conductance, Ca(2+)-activated K(+) channel of C. elegans, were obtained in a genetic screen for regulators of neurotransmitter release. Mutants were isolated by their ability to suppress lethargy of an unc-64 syntaxin mutant that restricts neurotransmitter release. Evoked postsynaptic currents were measured at the neuromuscular junction in both wild-type and mutants; the removal of SLO-1 greatly increases quantal content primarily by increasing duration of release. The selective isolation of slo-1 as the only ion channel mutant derived from a whole genomic screen to detect regulators of neurotransmitter release suggests that SLO-1 plays an important, if not unique, role in regulating neurotransmitter release (Wang, 2001).
The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. Cultured embryonic Xenopus neuromuscular junctions and simultaneous pre- and postsynaptic patch-clamp current-recording procedures were used to identify the major presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms elicit Na and K currents along with Ca2+ and Ca2+-activated K+ (KCa) currents. The onset of KCa current precede the peak of the action potential. The predominantly omega-CgTX GVIA-sensitive Ca current occurs primarily during the falling phase, but there is also significant Ca2+ entry during the rising phase of the action potential. The postsynaptic current begins a mean of 0.7 msec after the time of maximum rate of rise of the Ca2+ current. omega-CgTX also blocks KCa currents and transmitter release during an action potential, suggesting that Ca and KCa channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, KCa channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of KCa channels or the diffusion/removal of Ca2+ from sites of neurotransmitter release after an action potential. It is concluded that the currents carried by the N-type Ca2+ channels and K+ activated Ca2+ channels are functionally coactivated in presynaptic varicosities and coupled to transmitter release (Yazejian, 1997).
The kinetic and steady-state properties of macroscopic mslo (Slowpoke type channel) Ca-activated K+ currents were studied in excised patches from Xenopus oocytes. In response to voltage steps, the timecourse of both activation and deactivation, but for a brief delay in activation, can be approximated by a single exponential function over a wide range of voltages and internal Ca2+ concentrations ([Ca]i). Activation rates increase with voltage and with [Ca]i, and approach saturation at high [Ca]i. Deactivation rates generally decrease with [Ca]i and voltage, and approached saturation at high [Ca]i. Plots of the macroscopic conductance as a function of voltage (G-V) and the time constant of activation and deactivation shift leftward along the voltage axis with increasing [Ca]i. G-V relations could be approximated by a Boltzmann function with an equivalent gating charge which ranged between 1.1 and 1.8 e as [Ca]i varys between 0.84 and 1,000 microM. Hill analysis indicates that at least three Ca2+ binding sites can contribute to channel activation. Three lines of evidence indicate that there is at least one voltage-dependent unimolecular conformational change associated with mslo gating that is separate from Ca2+ binding. (a) The position of the mslo G-V relation does not vary logarithmically with [Ca]i. (b) The macroscopic rate constant of activation approaches saturation at high [Ca]i but remains voltage dependent. (c) With strong depolarizations mslo currents can be nearly maximally activated without binding Ca2+. These results can be understood in terms of a channel which must undergo a central voltage-dependent rate limiting conformational change in order to move from closed to open, with rapid Ca2+ binding to both open and closed states modulating this central step (Cui, 1997).
Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. The presence of voltage sensor rearrangements is demonstrated whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4-5 elementary charges (Stefani, 1997).
Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Channel activity was studied in cell-attached patches from hippocampal neurons and a unique specificity of coupling is reported. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains (Marrion, 1998).
As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L-type Ca2+ channels and ryanodine receptors (RyR) in cerebellar granule cells, an examination was carried out to see if such a coupling could activate a Ca2+-sensitive K+ channel, the big K+ (BK) channel, in cultured cerebellar granule cells. BK channels are specifically activation by group I mGluRs. Group I mGluRs stimulation of the basal BK channel activity is mimicked by caffeine and both effects were blocked by ryanodine and nifedipine. Interestingly, carbachol stimulates BK channel activity but through a pertussis toxin (PTX)-sensitive pathway that is independent of L-type Ca2+ channel activity. This report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by mobilizing an additional pathway involving RyR and L-type Ca2+ channels (Chavis, 1998).
The role of individual charged residues of the S4 region of a MaxiK channel (hSlo) in channel gating was investigated. Macroscopic currents induced by wild type (WT) and point mutants of hSlo were investigated in inside-out membrane patches of Xenopus laevis oocytes. Of all the residues tested, only neutralizations of Arg-210 and Arg-213 were associated with a reduction in the number of gating charges as determined using the limiting slope method. Channel activation in WT and mutant channels was interpreted using an allosteric model. Mutations R207Q, R207E, and R210N facilitate channel opening in the absence of Ca2+; however, this facilitation is not observed in the channels Ca2+-bound state. Mutation R213Q behaves similarly to the WT channel in the absence of Ca2+, but Ca2+ is unable to stabilize the open state to the same extent as it does in the WT. Mutations R207Q, R207E, R210N, and R213Q reduce the coupling between Ca2+ binding and channel opening when compared with the WT. Mutations L204R, L204H, Q216R, E219Q, and E219K in the S4 domain show a similar phenotype to the WT channel. It is concluded that the S4 region in the hSlo channel is part of the voltage sensor and that only two charged amino acid residues in this region (Arg-210 and Arg-213) contribute to the gating valence of the channel (Diaz, 1998).
Dehydrosoyasaponin-I (DHS-I) is a potent activator of high-conductance, calcium-activated potassium (maxi-K) channels. Interaction of DHS-I with maxi-K channels from bovine aortic smooth muscle was studied after incorporating single channels into planar lipid bilayers. Nanomolar amounts of intracellular DHS-I causes the appearance of discrete episodes of high channel open probability interrupted by periods of apparently normal activity. Statistical analysis of these periods reveals two clearly separable gating modes that likely reflect binding and unbinding of DHS-I. Kinetic analysis of durations of DHS-I-modified modes suggests DHS-I activates maxi-K channels through a high-order reaction. Average durations of DHS-I-modified modes increases with DHS-I concentration, and distributions of these mode durations contain two or more exponential components. In addition, dose-dependent increases in channel open probability from low initial values are high order with average Hill slopes of 2.4-2.9 under different conditions, suggesting at least three to four DHS-I molecules bind to maximally activate the channel. Changes in membrane potential over a 60-mV range appear to have little effect on DHS-I binding. DHS-I modified calcium- and voltage-dependent channel gating. 100 nM DHS-I causes a threefold decrease in concentration of calcium required to half maximally open channels. DHS-I shifts the midpoint voltage for channel opening to more hyperpolarized potentials with a maximum shift of -105 mV. 100 nM DHS-I has a larger effect on voltage-dependent compared with calcium-dependent channel gating, suggesting DHS-I may differentiate these gating mechanisms. A model specifying four identical, noninteracting binding sites, where DHS-I binds to open conformations with 10-20-fold higher affinity than to closed conformations, explains changes in voltage-dependent gating and DHS-I-induced modes. This model of channel activation by DHS-I may provide a framework for understanding protein structures underlying maxi-K channel gating, and may provide a basis for understanding ligand activation of other ion channels (Giagiacomo, 1998).
BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. In this study, the Ca2+ currents expressed in these cells are characterized and the Ca2+ channel subtypes are identified that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33%, 21%, 12%, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 muM nifedipine or Q-type Ca2+ channels by 2 muM Aga IVA reduces BK current activation by 77% and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 muM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduces BK current activation by only 11% and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminate activation of BK current during action potentials, whereas almost no effects are seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promotes activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels (Prakriya, 1999).
Recordings of the activity of the large conductance Ca2+-activated K+ (BK) channel from over 90% of inside-out patches excised from acutely dissociated hippocampal CA1 neurones reveal an inactivation process dependent upon the presence of at least 1 microM intracellular Ca2+. Inactivation is characterized by a sudden switch from sustained high open probability (Po) long open time behaviour to extremely low Po, short open time channel activity. The low Po state (mean Po, 0.001) consists of very short openings [time constant (tau), approximately 0.14 ms] and rare longer duration openings (tau, approximately 3.0 ms). Channel inactivation occurs with a highly variable time course being observed either prior to or immediately upon patch excision, or after up to 2 min of inside-out recording. Inactivation persists whilst recording conditions are constant. Inactivation is reversed by membrane hyperpolarization, the rate of recovery increasing with further hyperpolarization and higher extracellular K+. Inactivation is also reversed when the intracellular Ca2+ concentration is lowered to 100 nM and inactivation is permanently removed by application of trypsin to the inner patch surface. In addition, inactivation is perturbed by application of either tetraethylammonium ions or the Shaker (Sh)B peptide to the inner membrane face. During inactivation, channel Po is greater at hyperpolarized rather than depolarized potentials, which is partly the result of a greater number of longer duration openings. Depolarizing voltage steps (-40 to +40 mV) applied during longer duration openings produces only short duration events at the depolarized potential, yielding a transient ensemble average current with a rapid decay (tau, approximately 3.8 ms). These data suggest that hippocampal BK channels exhibit a Ca2+-dependent inactivation that is proposed to result from block of the channel by an associated particle. The findings that inactivation is removed by trypsin and prolonged by decreasing extracellular potassium suggest that the blocking particle may act at the intracellular side of the channel (Hicks, 1998).
Potassium channels play important roles in a wide variety of physiological processes. Although several genes encoding voltage-activated potassium channels have been analyzed at the molecular level, no calcium-activated potassium channel gene has yet been characterized in humans. In an effort to provide the foundation for functional analysis of such polypeptides, the cloning of mouse and human homologs of the Drosophila calcium-activated potassium channel gene, slowpoke, is reported. Both the human and mouse genes encode polypeptides that have more than 50% amino acid identifies with their Drosophila counterpart. In addition, like the Drosophila slowpoke gene, both the mouse and human genes generate multiple transcripts by alternative splicing. The human gene maps to chromosome 10 based on the results of polymerase chain reaction analysis of genomic DNA from human-hamster hybrid cell lines. Because calcium-activated potassium channels participate in wide variety of cellular functions including neuromuscular communication, secretion and cellular immunity, their continued analysis promises to have broad biological and medical significance (Pallanck, 1994).
Slo3 is a novel potassium channel abundantly expressed in mammalian spermatocytes. Slo3 represents a new and unique type of potassium channel regulated by both intracellular pH and membrane voltage. Slo3 is primarily expressed in testis in both mouse and human. Because of its sensitivity to both pH and voltage, Slo3 could be involved in sperm capacitation and/or the acrosome reaction, essential steps in fertilization where changes in both intracellular pH and membrane potential are known to occur. The protein sequence of mSlo3 (the mouse Slo3 homolog) is similar to Slo1, the large conductance, calcium- and voltage-gated potassium channel. These results suggest that Slo channels comprise a multigene family, defined by a combination of sensitivity to voltage and a variety of intracellular factors. Northern analysis from human testis indicates that a Slo3 homolog is present in humans and conserved with regard to sequence, transcript size, and tissue distribution. Because of its high testis-specific expression, pharmacological agents that target human Slo3 channels may be useful in both the study of fertilization as well as in the control or enhancement of fertility (Schreiber, 1998).
Na+-activated potassium channels (KNa) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. KNa is encoded by the rSlo2 gene (also called Slack), the mammalian ortholog of slo-2 in C. elegans. rSlo2, heterologously expressed, shares many properties of native KNa including activation by intracellular Na+, high conductance, and prominent subconductance states. In addition to activation by Na+, rSLO-2 channels are cooperatively activated by intracellular Cl-, similar to C. elegans SLO-2 channels. Since intracellular Na+ and Cl- both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis reveals that the SLO-2 current is a major component of the delayed rectifier. In C. elegans slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily (Yuan, 2003).
Large conductance, calcium-activated (BK) potassium channels play a central role in the excitability of cochlear hair cells. In mammalian brains, one class of these channels, termed Slo, is encoded by homologues of the Drosophila slowpoke gene. By homology screening with mouse Sla cDNA, a full-length clone (cSlo1) has been isolated from a chick's cochlear cDNA library, rSlol has greater than 90% identity with mouse Slo at the amino acid level, and is even better matched to a human brain Slo at the amino and carboxy termini. cSlol has none of the additional exons found in splice variants from mammalian brain. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to show expression of cSlal in the microdissected hair cell epithelium basilar papilla. Transient transfection of HIEK 293 cells demonstrates that cSlol encodes a potassium channel whose conductance averages 224 pS at +60 mV in symmetrical 140 mM K+. Macroscopic currents through cSlol channels are blocked by scorpion toxin or tetraethyl ammonium, and are voltage and calcium dependent. cSlol is likely to encode BK-type calcium-activated potassium channels in cochlear hair cells (Jiang, 1997).
Ionic fluxes across the sperm membrane have been shown to be important in the initiating process of sperm activation and gamete interaction; however, electrophysiological investigation of the ion channels involved has been precluded by the small size of the sperm, especially in mammalian species. In the present study sperm ion channels were expressed in Xenopus oocytes by injection of RNAs of spermatogenic cells isolated from the rat testes. The RNA-injected oocytes respond to ATP, a factor known to regulate sperm activation, with the activation of an outwardly rectifying whole-cell current which was dependent on K+ concentrations and inhibitable by K+ channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The ATP-induced current can be mimicked by a Ca2+ ionophore but suppressed by a Ca2+ chelator applied intracellularly, indicating a Ca2+ dependence of the current. Single-channel measurements on RNA-injected oocytes reveals channels of large conductance which can be blocked by CTX and TEA. Co-injection of germ cell RNAs with the antisense RNA for a mouse gene encoding slowpoke 'Maxi' Ca2+-activated K+ channels results in significant reduction of the ATP- and ionomycin-induced current. The expression of the 'Maxi' Ca2+-activated K+ channels in sperm collected from the rat epididymis was also confirmed by Western blot analysis. These results suggest that sperm possess Ca2+-activated K+ channels which may be involved in the process of sperm activation (Chan, 1998).
The Slack gene encodes a voltage-dependent K(+) channel that has a unitary conductance of approximately 60 pS. Evidence from heterologous expression studies suggests that Slack channel subunits can also combine with the Slo subunit to generate Ca(2+)-activated K(+) channels of larger conductances. Nonetheless, the function of Slack in the brain remains to be identified. An affinity-purified antibody was generated against the N-terminal of rat Slack, for biochemical and immunohistochemical studies. The antibody recognizes Slack in transiently transfected CHO cells both by immunocytochemistry and by Western blot analysis. The antibody also detects a single band in rat brain membranes. The localization of Slack in rat brain slices was then determined using the antibody. Most prominent Slack immunoreactivity occurs in the brainstem, in particular the trigeminal system and reticular formation, where very intense staining was found in both cell bodies and axonal fibers of associated nuclei. Labeling was also very strong in the vestibular and oculomotor nuclei. Within the auditory system, the medial nucleus of the trapezoid had a robust signal consistent with staining of the giant presynaptic terminals. Strong Slack immunoreactivity is present in the olfactory bulb, red nucleus, and deep cerebellar nuclei. There was labeling also in the thalamus, substantia nigra, and amygdala. The only cortical region in which Slack immunoreactivity is detected is the frontal cortex. The subcellular and regional distribution of Slack differs from that previously reported for the Slo channel subunit and suggests that Slack may also have an autonomous role in regulating the firing properties of neurons (Bhattacharjee, 2002).
Nine Ca2+-activated K+ channel isoforms from human brain have been cloned and expressed. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 termed hslo. RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca2+-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability (Tseng-Crank, 1994).
A family of alternatively spliced cDNAs has been cloned from the receptor epithelium of the chick cochlea. The cDNAs encode a Ca2+-activated K+ channel like those shown to help determine the resonant frequency of electrically tuned hair cells. PCRs using template RNAs from both tonotopically subdivide receptor epithelia and single hair cells demonstrate differential exon usage along the frequency axis of the epithelium at multiple splice sites of chicken Slowpoke type channels. Single hair cells express more than one splice variant at a given splice site. Since channel isoforms encoded by differentially spliced slo transcripts in other species are functionally heterogeneous, these data suggest that differential processing of slo transcripts may account, at least in part, for the systematic variation in hair-cell membrane properties along the frequency axis of electrically tuned auditory receptor epithelia (Navaratnam, 1997).
Cochlear frequency selectivity in lower vertebrates arises in part from electrical tuning intrinsic to the sensory hair cells. The resonant frequency is determined largely by the gating kinetics of calcium-activated potassium (BK) channels encoded by the slo gene. Alternative splicing of slo from chick cochlea generated kinetically distinct BK channels. Combination with accessory beta subunits slows the gating kinetics of alpha splice variants but preserves relative differences between them. In situ hybridization shows that the beta subunit is preferentially expressed by low-frequency (apical) hair cells in the avian cochlea. Interaction of beta with alpha splice variants could provide the kinetic range needed for electrical tuning of cochlear hair cells (Ramanathan, 1999).
The effect of ATP in the regulation of two closely related cloned mouse brain large conductance calcium- and voltage-activated potassium (BK) channel alpha-subunit variants, expressed in human embryonic kidney (HEK 293) cells, was investigated using the excised inside-out configuration of the patch-clamp technique. The mB2 BK channel alpha-subunit variant expressed alone is potently inhibited by application of ATP to the intracellular surface of the patch with an IC50 of 30 muM. The effect of ATP was largely independent of protein phosphorylation events as the effect of ATP is mimicked by the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) and the inhibitory effect of ATPgammaS is reversible. In contrast, under identical conditions, direct nucleotide inhibition is not observed in the closely related mouse brain BK channel alpha-subunit variant mbr5. Furthermore, direct nucleotide regulation is not observed when mB2 is functionally coupled to regulatory beta-subunits. These data suggest that the mB2 alpha-subunit splice variant could provide a dynamic link between cellular metabolism and cell excitability (Clark, 1999).
Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. A new model has been proposed where MaxiK channels have an additional transmembrane region (S0) at the N-terminus that confers beta subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and ''in vivo'' reconstitution assays, it is shown that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Hydrophobic segments S9-S10 in the C terminus are cytoplasmic and it is unequivocally demonstrated that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus (Meera, 1997).
The Slowpoke-related high-conductance Ca2+-activated K+ channel (mSlo) plays a vital role in regulating calcium entry in many cell types. mSlo channels behave like voltage-dependent channels, but their voltage range of activity is set by intracellular free calcium. The mSlo subunit has two parts: a 'core' resembling a subunit from a voltage-dependent K+ channel, and an appended 'tail' that plays a role in calcium sensing. Evidence is presented for a site on the tail that interacts with calcium. This site, the 'calcium bowl', is a novel calcium-binding motif that includes a string of conserved aspartate residues. Mutations of the calcium bowl fall into two categories: (1) those that shift the position of the G-V relation a similar amount at all Ca2+, and (2) those that shift the position of the G-V relation only at low Ca2+. None of these mutants alters the slope of the G-V curve. These mutant phenotypes are apparent in calcium ion, but not in cadmium ion, where mutant and wild type are indistinguishable. This suggests that the calcium bowl is sensitive to calcium ion, but insensitive to cadmium ion. The presence and independence of a second calcium-binding site is inferred because channels still respond to increasing levels of Ca2+ or [Cd2+], even when the calcium bowl is mutationally deleted. Thus a low level of activation in the absence of divalent cations is identical in mutant and wild-type channels, possibly because of activation of this second Ca2+-binding site (Schreiber, 1997).
A full length alpha-subunit of the Ca2+-activated K+ (BK) channel with an inactivating mutation in the C-terminus can complement a functional C-terminal fragment. Deletions and amino acid changes within the S8-S9 interdomain region were analyzed for their ability to allow complementation. Cys612 and His616 that are located in a region that contains two overlapping signature sequences, a immunoglobulin signature sequence and a heme binding domain, are essential for a functional channel. These two amino acid residues are also essential for complementation. The deletion of the PEST sequence does not affect the function of the BK channel; however, without the PEST sequence, complementation by a functional C-terminal fragments is no longer possible. The ability to complement a functional channel is restricted to the C-terminal fragment and requires that the complete alpha-subunit or the larger N-terminal fragment contains both, the immunoglobulin signature sequence the PEST sequence (Wood, 1997).
The 20 amino acid Shaker inactivation peptide blocks mSlo, a cloned calcium-dependent potassium channel. Changing the charge and degree of hydrophobicity of the peptide alters its blocking kinetics. A 'triple mutant' mSlo channel was constructed in which three amino acids (T256, S259, and L262), equivalent to those identified as part of the peptide's receptor site in the S4-S5 cytoplasmic loop region of the Shaker channel, were mutated simultaneously to alanines. These mutations produce only limited changes in the channel's susceptibility to block by a series of peptides of varying charge and hydrophobicity but do alter channel gating. The triple mutant channel shows a significant shift in its calcium-activation curve as compared with the wild-type channel. Analysis of the corresponding single amino acid mutations shows that mutation at position L262 causes the most dramatic change in mSlo gating. These results suggest that the three amino acids mutated in the mSlo S4-S5 loop may contribute to, but are not essential for, peptide binding. On the other hand, they do play a critical role in the channel's calcium-sensing mechanism (Sullivan, 1997).
The high-conductance Ca2+-activated K+ (maxi-K) channel from bovine tracheal smooth muscle was purified to apparent homogeneity by a combination of conventional chromatographic techniques and sucrose density gradient centrifugation. Fractions with the highest specific activity for binding of monoiodotyrosine charybdotoxin, [125I]ChTX, were enriched approximately 2000-fold over the initial digitonin-solubilized material up to a specific activity of 1 nmol/mg protein. Silver staining after SDS-polyacrylamide gel electrophoresis of the fractions from the last step of the purification indicates that binding activity is correlated with a major component of the preparation that displays an apparent molecular weight of 62,000. Labeling the same preparation with 125I-Bolton-Hunter reagent reveals the existence of both 62 (alpha)- and 31 (beta)-kDa subunits, in an apparent stoichiometry of 1:1, comigrating with binding activity. The beta subunit is heavily glycosylated. Deglycosylation studies indicate that the beta subunit represents the protein to which [125I]ChTX is covalently incorporated in the presence of the bifunctional cross-linking reagent disuccinimidyl suberate. Binding of [125I]ChTX to the purified ChTX receptor displayed the same pharmacological profile that has been found previously for toxin binding to native membranes, including inhibition by iberiotoxin, limbatustoxin, tetraethylamonium, potassium, cesium, and barium. The purified preparation was reconstituted into liposomes which were then fused with artificial lipid bilayers. Single channels were readily observed with a conductance of 235 picosiemens in 150 mM KCl that displayed selectivity for potassium over chloride and that were blocked by ChTX. The open probability of these channels was increased by depolarizing membrane potentials and by raising the internal calcium concentration. These data suggest that the maxi-K channel purified from tracheal smooth muscle is composed of two subunits (Garcia-Calvo, 1994).
Coexpression of alpha and beta subunits of the high conductance Ca2+-activated K+ (maxi-K) channel leads to a 50-fold increase in the affinity for 125I-charybdotoxin (125I-ChTX) as compared with when the alpha subunit is expressed alone. To identify those residues in the beta subunit that are responsible for this change in binding affinity, Ala scanning mutagenesis was carried out along the extracellular loop of beta, and the resulting effects on 125I-ChTX binding were determined after coexpression with the alpha subunit. Mutagenesis of each of the four Cys residues present in the loop causes a large reduction in toxin binding affinity, suggesting that these residues could be forming disulfide bridges. The existence of two disulfide bridges in the extracellular loop of beta was demonstrated after comparison of reactivities of native beta and single-Cys-mutated subunits to N-biotin-maleimide. Negatively charged residues in the loop of beta, when mutated individually or in combinations, has no effect on toxin binding with the exception of Glu94, whose alteration modifies kinetics of ligand association and dissociation. Further mutagenesis studies targeting individual residues between Cys76 and Cys103 indicate that four positions, Leu90, Tyr91, Thr93, and Glu94 are critical in conferring high affinity 125I-ChTX binding to the alpha.beta subunit complex. Mutations at these positions cause large effects on the kinetics of ligand association and dissociation, but they do not alter the physical interaction of beta with the alpha subunit. All these data, taken together, suggest that the large extracellular loop of the maxi-K channel beta subunit has a restricted conformation. Moreover, they are consistent with the view that four residues appear to be important for inducing an appropriate conformation within the alpha subunit that allows high affinity ChTX binding (Hanner, 1998).
Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, it was found that the beta subunit increases open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit is not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibits transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle have bursting kinetics similar to channels expressed from alpha subunits alone (Nimigean, 1999).
Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcriptional regulation of maxiK channels, the promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel complex were characterized. Maximum promoter activity (up to 12.3-fold over control) occurs between nucleotides -567 and -220 for the alpha subunit (hSlo) gene. The minimal promoter is GC-rich with 5 Sp-1 binding sites and several TCC repeats. Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1, PEA-3, Myo-D, and E2A, are observed in the 5'-flanking sequence. Additionally, a CCTCCC sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located 27 bp upstream of the TATA-like sequence, a location identical to that found in the SM1/2 5'-flanking region. However, the promoter directs equivalent expression when transfected into smooth muscle and other cell types. Analysis of the hSlo beta subunit 5'-flanking region revealed a TATA box at position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity is not observed. The lack of tissue-specific promoter activity, particularly the finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the promoters of the two genes suggests that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences (Dhulipala, 1999).
Voltage- and Ca2+-sensitive K+ (MaxiK) channels play key roles in controlling neuronal excitability and vascular tone. Human and rodent genes for the modulatory beta subunit, KCNMB1, have been cloned and analyzed. The human and mouse beta-subunit genes are approximately 11 and approximately 9 kb in length, respectively, and have a four exon-three intron structure. Primer extension assay localized the transcription initiation site at 442 (human) or 440 (mouse) bp upstream of the translation initiation codon, agreeing with the transcript size in Northern blots. Both genes have a TATA-less putative promoter region, with a transcription initiator-like region, and motifs characteristic of regulated promoters, including muscle-specific enhancing factors-1 and -2. Consistent with a tissue-specific expression of KCNMB1, regulated at the transcriptional level, beta-subunit transcripts are abundant in smooth muscle and heart, but scarce in lymphatic tissues, brain, and liver. Expressed rat and mouse beta subunits increase the apparent Ca2+ sensitivity of the human MaxiK channel alpha subunit (Jiang, 1999).
Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. A transmembrane beta subunit (beta2) is reported that yields inactivating MaxiK currents on coexpression with the pore-forming alpha subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the beta2 subunit abolishes inactivation of the alpha subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle beta1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the beta2 subunit causes inactivation of the MaxiK channel alpha subunit by occluding its K+-conducting pore resembling the inactivation caused by the 'ball' peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different beta subunits (Wallner, 1999).
The human large conductance, calcium-activated potassium (maxi-K) channel (alpha and beta subunits) and beta2-adrenergic receptor genes were coexpressed in Xenopus oocytes in order to study the mechanism of beta-adrenergic modulation of channel function. Isoproterenol and forskolin increase maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and both channel subunits by 33% +/- 5% and 35% +/- 8%, respectively, without affecting current activation or inactivation. The percentage of stimulation by isoproterenol and forskolin is not different in oocytes coexpressing the alpha and beta subunits versus those expressing the only the alpha subunit, suggesting that the alpha subunit is the target for regulation. The stimulatory effect of isoproterenol is almost completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK) regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide has little effect, indicating that cellular coupling of beta2-adrenergic receptors to maxi-K channels involves endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation sites (serine 869) on the alpha subunit almost completely inhibits beta-adrenergic receptor/channel stimulatory coupling, whereas forskolin still stimulates currents moderately (16% +/- 4%). These data demonstrate that physiological coupling between beta2 receptors and maxi-K channels occurs by the cAMP-PK mediated phosphorylation of serine 869 on the alpha subunit on the channel (Nara, 1998).
The cloned BK channel alpha subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOalpha cells) or in combination with the auxiliary beta subunit (CHOalpha+beta cells). Basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity were studied. Coexpression of alpha and beta subunits enhance the Ca2+ and voltage sensitivity of the BK channel, and decrease the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, are independent of the beta subunit. The cGMP kinase Ialpha and cAMP kinase fail to affect BK channel activity in CHOalpha and CHOalpha+beta cells at different Ca2+i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women respond to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NPo), respectively. These effects can be reversed by alkaline phosphatase and remain unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increase NPo 2-fold and 3.5-fold, respectively. This stimulation is enhanced rather than reversed by alkaline phosphatase and is abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase is observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study (Zhou, 1998).
Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I were used. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increases the macroscopic currents. This increase in current is due to an increase in the channel voltage sensitivity by approximately 20 mV and is reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increases their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels (Alioua, 1999).
The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. The mechanism of calcium gating has been studied and SK channels are found not to be gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. These data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein (Xia, 1998a).
Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which are sensitive to both membrane potential and intracellular calcium. A potassium channel gene called Slack has been isolated has been isolated from the rat that is abundantly expressed in the nervous system. Slack channels rectify outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium. However, when Slack is co-expressed with Slo, channels with pharmacological properties and single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. These findings indicate that some intermediate-conductance channels in the nervous system may result from an interaction between Slack and Slo channel subunits (Joiner, 1998).
Voltage- and calcium-dependent BK channels regulate calcium-dependent cellular events such as neurotransmitter release by limiting calcium influx. Their plasma membrane abundance is an important factor in determining BK current and thus regulation of calcium-dependent events. This study shows that in C. elegans, ERG-28 (see Drosophila CG17270), an endoplasmic reticulum (ER) membrane protein, promotes the trafficking of SLO-1 (see Drosophila slo) BK channels from the ER to the plasma membrane by shielding them from premature degradation. In the absence of ERG-28, SLO-1 channels undergo aspartic protease DDI-1-dependent degradation, resulting in markedly reduced expression at presynaptic terminals. Loss of erg-28 suppresses phenotypic defects of slo-1 gain-of-function mutants in locomotion, neurotransmitter release, and calcium-mediated asymmetric differentiation of the AWC olfactory neuron pair, and confer significant ethanol-resistant locomotory behavior, resembling slo-1 loss-of-function mutants, albeit to a lesser extent. These data thus indicate that the control of BK channel trafficking is a critical regulatory mechanism for synaptic transmission and neural function (Oh, 2017).
Mutations in the cereblon (CRBN) gene cause human intellectual disability, one of the most common cognitive disorders. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. This study investigated the role of CRBN in synaptic function and animal behavior using male mouse and Drosophila models (ohgata). Crbn knockout (KO) mice showed normal brain and spine morphology as well as intact synaptic plasticity; however, they also exhibited decreases in synaptic transmission and presynaptic release probability exclusively in excitatory synapses. Notably, presynaptic function was impaired not only by loss of CRBN expression, but also by expression of pathogenic CRBN mutants (human R419X mutant and Drosophila G552X mutant). BK channel (Slowpoke in Drosophila) blockers paxilline and iberiotoxin reversed this decrease in presynaptic release probability in Crbn KO mice. In addition, paxilline treatment also restored normal cognitive behavior in Crbn KO mice. These results strongly suggest that increased BK channel activity is the pathological mechanism of intellectual disability in CRBN mutations (Choi, 2018).
Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre-motor circuit activity. This study utilized the fruit fly, Drosophila, to study a PxD linked to a gain-of-function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel (Drosophila homolog: Slowpoke). This study aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. Attempts were made to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre-motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption. A knock-in Drosophila model was generated using homologous recombination. Electrophysiological recordings and calcium-imaging were used to assess AP shape, neurotransmission, and the activity of the larval pre-motor central pattern generator (CPG). Video-tracking and automated systems were used to measure movement, and developed a genetic method to limit BK channel expression to defined circuits. Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. Calcium-dependent reductions were identified in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, aberrant locomotion and dyskinesia-like movements were identified in adult model flies, and the impact of GOF BK channels on movement was partially mapped to cholinergic neurons. This model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs (Kratschmer, 2021).
Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre-motor circuit activity. This study utilized the fruit fly, Drosophila, to study a PxD linked to a gain-of-function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel (Drosophila homolog: Slowpoke). This study aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. Attempts were made to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre-motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption. A knock-in Drosophila model was generated using homologous recombination. Electrophysiological recordings and calcium-imaging were used to assess AP shape, neurotransmission, and the activity of the larval pre-motor central pattern generator (CPG). Video-tracking and automated systems were used to measure movement, and developed a genetic method to limit BK channel expression to defined circuits. Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. Calcium-dependent reductions were identified in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, aberrant locomotion and dyskinesia-like movements were identified in adult model flies, and the impact of GOF BK channels on movement was partially mapped to cholinergic neurons. This model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs (Kratschmer, 2021).
Search PubMed for articles about Drosophila Slowpoke
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date revised: 20 May 2018
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