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Gene name - slowpoke Synonyms - 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 |
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 (~37ƒ) 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 37ƒ, 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 30ƒ. 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).
Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca2+-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca2+-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca2+-activated K+ channels (Atkinson, 1991).
The pore-forming alpha subunit of large conductance voltage- and Ca2+-sensitive K (MaxiK) channels is regulated by a beta subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming alpha subunit necessary for beta-subunit modulation, chimeric constructs were made between a human MaxiK channel and the Drosophila homolog. The construct is insensitive to beta-subunit modulation. The topology of the alpha subunit was analyzed. A comparison of multiple sequence alignments with hydrophobicity plots reveals that MaxiK channel alpha subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, a new model is proposed where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homolog transfers beta-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function (Wallner, 1996).
Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds that belongs to the Kunitz family of serine proteinase inhibitors. BPTI is an extremely potent inhibitor of trypsin, but it also specifically binds to various active and inactive serine proteinase homologs with KD values that range over eight orders of magnitude. An interaction of BPTI at an intracellular site has been described that results in the production of discrete subconductance events in large conductance Ca2+ activated K+ channels. BPTI binds to a site on the KCa channel protein that structurally resembles a serine proteinase. One line of evidence includes the finding that the complex of BPTI and trypsin, in which the inhibitory loop of BPTI is masked by interaction with trypsin, is completely ineffective in the production of substate events in the KCa channel. To further investigate this notion, a sequence analysis was performed of the alpha-subunit of cloned slowpoke KCa channels from Drosophila and mammals. This analysis suggests that a region of approximately 250 residues near the COOH terminus of the KCa channel is homologous to members of the serine proteinase family, but is catalytically inactive because of various substitutions of key catalytic residues. The sequence analysis also predicts the location of a Ca2+-binding loop that is found in many serine proteinase enzymes. It is hypothesized that this COOH-terminal domain of the slowpoke KCa channel adopts the characteristic double-barrel fold of serine proteinases, is involved in Ca2+-activation of the channel, and may also bind other intracellular components that regulate KCa channel activity (Moss, 1997).
date revised: 16 May 99
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