InteractiveFly: GeneBrief

small conductance calcium-activated potassium channel: Biological Overview | References

Gene name - small conductance calcium-activated potassium channel

Synonyms -

Cytological map position - 4F5-4F9

Function - transmembrane channel

Keywords - potassium channel - negatively regulates nociception - interacts with calmodulin, which acts as a Ca2+ sensor - regulates synaptic excitation in the postsynapse of the NMJ - contributes to photoreceptor performance by mediating sensitivity control at the first visual network - negatively regulates the acquisition of short-term memory

Symbol - SK

FlyBase ID: FBgn0029761

Genetic map position - chrX:5,339,792-5,405,142

Classification - SK_channel: Calcium-activated SK potassium channel, Calmodulin binding domain, Ion channel

Cellular location - surface transmembrane

NCBI link: EntrezGene, Nucleotide, Protein
SK orthologs: Biolitmine
Recent literature
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.
Castelli, L. M., Cutillo, L., Souza, C. D. S., Sanchez-Martinez, A., Granata, I., Lin, Y. H., Myszczynska, M. A., Heath, P. R., Livesey, M. R., Ning, K., Azzouz, M., Shaw, P. J., Guarracino, M. R., Whitworth, A. J., Ferraiuolo, L., Milo, M. and Hautbergue, G. M. (2021). SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis. Mol Neurodegener 16(1): 53. PubMed ID: 34376242
Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. This study investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of a gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. This study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. This study also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.

In Drosophila larvae, Class IV sensory neurons respond to noxious thermal stimuli and provoke heat avoidance behavior. Previously, work has shown that the activated neurons displayed characteristic fluctuations of firing rates, which consisted of repetitive high-frequency spike trains and subsequent pause periods, and it has been proposed that the firing rate fluctuations enhanced the heat avoidance (Terada, 2016). This study further substantiates this idea by showing that the pause periods and the frequency of fluctuations are regulated by small conductance Ca(2+)-activated K(+) (SK) channels, and the SK knockdown larvae display faster heat avoidance than control larvae. The regulatory mechanism of the fluctuations in the Class IV neurons resembles that in mammalian Purkinje cells, which display complex spikes. Furthermore, the results suggest that such fluctuation coding in Class IV neurons is required to convert noxious thermal inputs into effective stereotyped behavior as well as general rate coding (Onodera, 2017).

Animals sense diverse environmental inputs, including noxious ones, by using specific sensory organs. In principle, sensory neurons convert the intensity of stimuli into the magnitude of firing rates upon sensory transduction. For instance, mammalian C-fiber nociceptors convert gentle touch stimuli into relatively low firing rates, whereas injurious forces elicit higher rates. The 'rate coding' is valuable for sensory transduction, particularly with regard to stimulus intensity; however, the firing rate has an intrinsic upper limit because interspike intervals (ISIs) cannot be shorter than refractory periods, when the membrane is unable to respond to another stimulus. This implies that firing rates should saturate at high intensities, at which point the sensory inputs are no longer converted properly in an intensity-to-firing rate correspondence. Therefore, it is assumed that some sensory neurons may use other coding mechanisms that are employed in the central nervous system (Onodera, 2017).

In Drosophila larvae, Class IV dendritic arborization neurons (Class IV neurons) are primary nociceptive neurons that respond to multiple stimuli, including high temperature, strong mechanical force, and short-wavelength light. When the neurons are activated by noxious thermal stimuli, for instance, their sensory transduction provokes heat avoidance behavior where larvae rotate around the long body axis in a corkscrew-like manner. A large number of genes responsible for the neuronal activation were identified by evaluating behavioral phenotypes and monitoring Ca2+ dynamics in mutant strains; however, there have been few studies which have investigated the coding mechanism of the nociception by recording electrical activity (Onodera, 2017).

A previous study built a measurement system using a 1460 nm infrared (IR) laser as a local heating device and found that Class IV neurons were found to responded to noxious thermal stimuli with evoked characteristic fluctuations of firing rates, which consisted of repetitive high-frequency spike trains and subsequent quiescent periods (Terada, 2016). The occurrence of such 'burst-and-pause' firing patterns was coordinated with large Ca2+ increments over the entire dendritic arbors (designated as dendritic Ca2+ transients here) and was mediated by L-type voltage-gated Ca2+ channels (VGCCs). Knocking down L-type VGCCs in neurons abolished the burst-and-pause firing patterns, and the knockdown larvae displayed delayed heat avoidance behavior. Therefore, it was hypothesized that the burst-and-pause firing patterns should be output signals transducing high intensity stimuli and provoking the robust avoidance behavior. However, the regulatory mechanism of the firing patterns remained unclear because L-type VGCCs produce depolarizing currents but not hyperpolarizing ones, which should underlie 'pause' periods. This study showed that the pause period and the number of the burst-and-pause firing patterns are regulated by small conductance Ca2+-activated K+ (SK) channels, and that SK knockdown larvae display relatively fast heat avoidance. Furthermore, this study showed that one of the downstream neurons dramatically changes the response to two optogenetic activations of the Class IV neurons which have distinct numbers of burst-and-pause firing patterns. These findings strengthen the hypothesis and suggest that the 'fluctuation coding' is required to convert high intensities of noxious thermal stimuli into the robust, appropriate avoidance behavior as well as general rate coding (Onodera, 2017).

Although the increased number of unconventional spikes (USs) in SK knockdown neurons may initially seem counterintuitive, it can be explained comprehensively by two states of SK channels, at low and high activation levels: (1) Before USs occur, most SK channels are in the steady state because the Ca2+/calmodulin association is restricted at low [Ca2+]i, and the SK current slightly inhibits the incidence of firings during burst periods. Therefore, SK knockdown attenuates the inhibition of firings, which raises the occurrence rate of USs. (2) In contrast, after USs occur with dendritic Ca2+ transients, the channels are shifted to the activation state by high [Ca2+]i, and the current greatly promotes after-hyperpolarization, which generates the pause periods. Thus, the knockdown dramatically decreases the pause periods, which shortens the time requiring one burst-and-pause firing pattern. Due to the two impacts on firings, the US number per unit time would be expected to increase upon SK knockdown (Onodera, 2017).

It was hypothesized that the burst-and-pause firing patterns in Class IV neurons are regulated by functional coordination between L-type VGCCs and SK channels as follows: (1) Thermosensitive channels including dTrpA1 and Painless are activated by high-temperature stimulation and elicit the initial membrane depolarization in the dendritic arbors. (2) Once the membrane potential of soma exceeds a certain threshold by the prolonged stimulation, the neurons evoke action potentials and then increase firing rates with the intensity of stimulation ('rate coding'). (3) When L-type VGCCs in the dendritic arbors are activated by the high-order depolarization, they induce a large Ca2+ influx, which rapidly activates SK channels. (4) The activated SK channels produce a hyperpolarizing current, thereby generating the pause periods ('fluctuation coding'). It is also suggested that other K+ channels may slightly contribute to the generation of pauses, because the pause periods were not completely abolished in SK knockdown neurons. Although the other candidate channels, such as Sh and Shal, are not activated by the [Ca2+]i rise, most of them are voltage-dependent and hence hyperpolarize the membrane potential to some degree after depolarization, regardless of dendritic Ca2+ influx. Because the hyperpolarization suppresses the probability of firing, including US, the knockdown of those channels should lead to the increment of the US number (Onodera, 2017).

In the mammalian cerebellar cortex, climbing fiber inputs evoke complex spikes of Purkinje cells, which induce a dendritic Ca2+ influx through Ca2+ spikes and subsequent pauses. The pause periods of post-complex spikes are regulated by dendritic Ca2+ spikes, which are dependent on P/Q-type VGCCs, and are modulated by after-hyperpolarization, which is largely dependent on SK2 channels (Grasselli, 2016). Considering these observations, the regulatory mechanism of complex spikes is remarkably similar to that of burst-and-pause firing patterns in Class IV neurons (Onodera, 2017).

In principle, sensory neurons convert the intensity of stimuli into the magnitude of firing rates. This form of rate coding also occurs in Class IV neurons at relatively low temperatures, and it is mediated by thermosensitive channels and many types of voltage-gated ion channels. At higher temperature, however, L-type VGCCs and SK channels modulate the firing, transitioning from continuous high-frequency patterns into burst-and-pause patterns. Thus, it is proposed that the firing-rate-fluctuation coding allows sensory neurons to transmit strong stimuli not covered in rate coding, thereby provoking robust avoidance behavior (Onodera, 2017).

The Drosophila small conductance calcium-activated potassium channel negatively regulates nociception

Inhibition of nociceptor activity is important for the prevention of spontaneous pain and hyperalgesia. To identify the critical K(+) channels that regulate nociceptor excitability, a forward genetic screen was performed using a Drosophila larval nociception paradigm. Knockdown of three K(+) channel loci, the small conductance calcium-activated potassium channel (SK), seizure, and tiwaz, causes marked hypersensitive nociception behaviors. In more detailed studies of SK, this study found that hypersensitive phenotypes can be recapitulated with a genetically null allele. Optical recordings from nociceptive neurons showed a significant increase in mechanically activated Ca(2+) signals in SK mutant nociceptors. SK is expressed in peripheral neurons, including nociceptive neurons. Interestingly, SK proteins localize to axons of these neurons but are not detected in dendrites. These findings suggest a major role for SK channels in the regulation of nociceptor excitation and are inconsistent with the hypothesis that the important site of action is within dendrites (Walcott, 2018).

The sensation of pain is important for avoiding exposure to noxious environmental stimuli that have the potential to cause tissue damage. These stimuli are detected by nociceptors, which are the primary sensory neurons that detect noxious mechanical, noxious chemical, and/or noxious temperatures. Transduction of noxious thermal, mechanical, and chemical stimuli is initiated by sensory receptor ion channels, which depolarize the sensory neuron plasma membrane and trigger action potentials. In the absence of such stimuli, healthy nociceptors remain relatively silent, with little spontaneous activity due to the action of potassium (K+) channels and chloride (Cl-) channels, which oppose depolarizing sodium (Na+) and calcium (Ca2+) currents. Despite their importance in keeping nociceptive neurons silent, the identity of the K+ channels that play the most critical roles in negatively regulating nociceptor excitability remains largely undetermined (Walcott, 2018).

To identify these critical channels, a forward genetic screen was conducted using a modified Drosophila larval nociception paradigm that was optimized for detecting hypersensitive nociception phenotypes (Walcott, 2018).

A collection of transgenic RNAi strains from the Vienna Drosophila Resource Center (VDRC) and the Transgenic RNAi Project (TRiP) allow for in vivo tissue-specific gene silencing under control of the Gal4/UAS system. 53 UAS-inverted repeat (UAS-IR) RNAi lines in these collections were identifed that targeted 34 K+ channels with few predicted off-target effects. All known Drosophila K+ channels are represented in the assembled collection (Walcott, 2018).

The effects were investigated of knocking down the K+ channels under control of the GAL4109(2)80;UAS-Dicer2 (md-Gal4;UAS-Dicer2) driver strain. This strain drives UAS transgene expression in the class I, II, III, and IV md neurons. Evidence suggests that the major nociceptive function is mediated by the class IV md neurons, but class II and class III neurons are also involved (Hwang, 2007, Hu, 2017). The use of UAS-Dicer2 in the driver strain results in more efficient gene silencing. To perform the screen, the md-Gal4;UAS-Dicer2 driver strain was crossed to each of the 53 UAS-RNAi strains targeting the K+ channels and the nocifensive escape locomotion (NEL) response latency of the larval progeny stimulated with a 42°C heat probe were measured. The crossed progeny from UAS-RNAi lines targeting three distinct K+ channel subunits showed a significantly more rapid response relative to the genetic background control strain: the small conductance calcium-activated potassium channel (SK), the seizure channel (in the ether-a-gogo family), and the tiwaz gene (encodes a protein with homology to the potassium channel tetramerization domain). Although phenotypes were not observed for other tested K+ channels, this method for RNAi is prone to false negatives, so the screen cannot rule out potential involvement for other channels. To test whether the effects of the RNAi were specific to the nociceptive class IV sensory neurons, animals were tested expressing UAS-RNAi targeting these three candidates under control of ppk-Gal4;UAS-Dicer2. The hypersensitive responses persisted in SK-RNAi and seizure-RNAi animals (Walcott, 2018).

A prior pharmacological study on mammalian sensory neurons suggested an SK-mediated pathway for nociceptor excitability; however, the cellular role of this ion channel specifically in nociception remains largely unexplored and has not been verified with genetic mutants. The mammalian genome contains three genes that encode SK channel subunits, while the Drosophila genome encodes only a single SK locus on the X chromosome that is 60 kb in length and is predicted to encode at least 14 distinct transcripts. The Drosophila SK locus has been found to mediate, a slow Ca2+-activated K+ current in photoreceptor neurons and muscle as well as playing a role in learning and memory (Abou Tayoun, 2011; Abou Tayoun, 2012; Gertner, 2014). To further investigate the function of SK, a DNA null mutant was generated by deleting the gene with Flippase (FLP) and FLP recombination target (FRT)-containing transposons. Consistent with the hypothesis that SK is an important negative regulator of nociception, SK null mutants (ΔSK) showed a pronounced hypersensitive response at 42°C. These animals showed an average response latency to a 42°C stimulus of 3.2 s, which was significantly faster than the control background strain response of 6.2 s (Walcott, 2018).

To confirm that loss of SK was responsible for the nociception defect, a genetic rescue experiment was performed through transgenic insertion of an ~80-kb bacterial artificial chromosome (BAC) that covered the entire SK locus. The BAC transgenic flies were crossed into the SK genetic mutant background to create rescue animals containing either one or two copies of the BAC transgene covering the SK genomic region. The nociception hypersensitivity phenotype was fully reverted in rescue animals containing two copies of the BAC transgene. Interestingly, animals heterozygous for the SK mutation exhibit hypersensitivity to noxious heat but to a lesser degree than homozygotes (4.8 s). Note that testing of heterozygotes can only be performed in female larvae as SK is located on the X chromosome (thus, female larvae were used in all experiments to allow for consistent comparisons). One copy of the BAC rescue transgene provides only partial rescue of the hypersensitivity phenotype (5.0 s) but two copies fully rescued. These data combined support a dosage-sensitive, semi-dominant thermal nociception defect for SK mutants that requires two copies of the BAC transgene for phenotypic full rescue (Walcott, 2018).

To test for a nociceptor-specific requirement for SK, the SK-M transcript was expressed under control of the ppk-GAL4 driver in the SK mutant background. This manipulation fully rescued the hypersensitive nociception phenotype of the SK mutant animals, confirming the site of action for SK in the nociceptor neurons. SK-M is one of eight long protein isoforms that are annotated on Flybase, and there are an additional six predicted short isoforms. As well, a cDNA was cloned for a transcript encoding a seventh short SK isoform. Unlike the current experiments with SK-M, expression of the SK-V transcript in nociceptors did not result in a rescue of the hypersensitive mutant phenotype. These experiments suggest that long SK isoforms may be more important than short isoforms for suppressing the thermal sensitivity of nociceptors (Walcott, 2018).

The elaborately branched class IV neurons function as polymodal nociceptors, playing a role in both thermal (≥39°C) and mechanical nociception (≥30 mN). Channels expressed in class IV neurons such as such Painless and dTRPA1 are required for both thermal and mechanical nociception, while Pickpocket, Balboa/PPK26, and Piezo have more specific roles in mechanical nociception. Interestingly, SK mutant larvae showed enhanced nocifensive responses to a 30-mN mechanical stimulus compared to parental strain animals. With this stimulus, ΔSK mutant animals rolled in response to the noxious force stimuli in 58% of trials, while control animals responded in only 31%. As with thermal nociception, replacing SK in the genome by BAC transgene restored the mechanical nociception response to wild-type levels with 38% of BAC rescue animals responding to the 30-mN stimulus. However, the mechanical nociception phenotype was less sensitive to dosage. Animals heterozygous for the ΔSK mutation as well as animals containing one copy of the BAC rescue transgene respond similarly to wild-type animals (44% and 39%, respectively). As with thermal nociception, UAS-SK-M expressed under control of the ppk-GAL4 driver fully rescued the SK mutant mechanical nociception phenotype (Walcott, 2018).

To determine whether SK disruption affects mechanosensation in general, SK mutant larvae in an established gentle touch assay. Gentle touch responses in SK mutants appeared normal. Thus, the somatosensory effects of SK were more specific to the nociception pathway, regulating nociceptor activity both in response to noxious thermal and mechanical stimuli (Walcott, 2018).

Next, optical recordings were performed from control (Exelixis isogenic white) and SK mutant larvae expressing the genetically encoded Ca2+ indicator, GCaMP3.0, under the control of the nociceptor-specific driver, ppk-Gal4. In this filleted larval preparation, the md neurons expressing GCaMP3.0 were imaged through the transparent cuticle using high-speed, time-lapse confocal microscopy while stimulated with a 50-mN probe. ppk-Gal4-expressing neurons imaged in this preparation showed rapidly increasing GCaMP3.0 signals during the initial application of force and this signal rapidly declined. In SK mutant animals, the peak calcium response (measured at the cell soma) was significantly increased relative to wild-type, and the signal remained elevated above the baseline for several seconds following the mechanical stimulus. Restoration of SK-M to the mutant background rescued the elevated peak response but did not fully suppress the prolonged signal seen in the mutant. Thus, although the SK-M isoform can rescue behavioral phenotypes and peak calcium responses in class IV neurons, it is possible that one or more of the 13 other isoforms is required for complete restoration of wild-type responses in this Ca2+ imaging assay (Walcott, 2018).

To evaluate the expression of SK, a transgenic Drosophila strain from the Minos-mediated integration cassette (MiMIC) collection. A MiMIC element inserted in the proper orientation into the 5' non-coding intron of SK long isoform transcripts should express EGFP in the native pattern of SK (i.e., at endogenous levels in appropriate tissues). EGFP expression was observed in the larval peripheral nervous system in a subset of type I and type II sensory neurons that included the class IV md neurons. These results reveal that transcripts encoding long-isoform SK proteins are endogenously expressed in the nociceptors and provide additional validation of the tissue-specific UAS-SK-M rescue experiments that restored normal nociception responses to SK mutant larvae (Walcott, 2018).

The potassium currents mediated by SK channels in mammalian neurons make an important contribution to the afterhyperpolarization (AHP) of action potentials. However, the precise subcellular function of SK channels is thought to vary depending on the subcellular compartment where it resides. SK proteins have been found in somatic or dendritic compartments in some cell types, and in axons or presynaptic compartments in others. Thus, it was desirable to identify the subcellular compartment containing SK channels in the nociceptive md neuron (Walcott, 2018).

Using a previously generated anti-SK antibody (Abou Tayoun, 2011), subcellular localization of the SK-M protein was observed used in the nociceptor-specific rescue experiment. Surprisingly, it was found that the rescuing SK-M protein was clearly detectable in the class IV axons and soma but only weakly detectable in proximal dendrites . This was consistent with staining in wild-type animals, where it was possible to detect SK protein in axons of sensory neurons but it was not possible to detect any expression in the dendrites or soma of class IV neurons. Note that, because the SK antibody detects sensory neuron axons of multiple types in the dorsal sensory neuron cluster, it was often impossible to unambiguously assign the axonal staining observed in wild-type animals to the ddaC class IV axon. However, the anti-SK staining was completely eliminated in null mutant animals confirming specificity of the antibody (Walcott, 2018).

These results raised the possibility that SK proteins are required in axons and not in dendrites for nociception. The anti-SK antibody was raised against a purified SK fragment fusion protein largely composed of the N-terminal domain but the exact epitope it detects is not known (Abou Tayoun, 2011). Thus, it remained possible that isoforms of SK not detected by this antibody might localize to dendrites. Therefore, in order to detect as many SK protein isoforms as possible, and at their endogenous expression levels, CRISPR-mediated homologous repair was used at the SK locus. The inserted V5 epitope tag is encoded by an exon present in 13 of the 14 known SK transcripts (with the exception of the SK-J) located immediately downstream of the sequence encoding the SK calmodulin-binding domain. Interestingly, anti-V5 directed immunofluorescence in animals with the genomic modification was present in the axons of peripheral sensory neurons. Specifically, strong labeling was observed of axons of a subset of type I (external sensory [es] and chordotonal) neurons and type II md neurons, including the class IV. The latter could be unambiguously identified in the v'ada class IV cell because its axon does not bundle with other axons prior to entering the nerve. Interestingly SK::V5 proteins concentrate within a proximal compartment of axons in class IV sensory neurons but were not detected in nociceptor axon terminals in the CNS or in sensory dendrites of the nociceptive neuron arbor. The subcellular distribution of SK::V5 proteins along the axon of sensory neurons corroborated labeling of similar neuronal structures with anti-SK antibodies targeting long isoforms of SK (Abou Tayoun, 2011). Note that the SK-J protein contains the N-terminal domain that was used for raising the anti-SK antibody, making it unlikely that it localizes to dendrites. Thus, the current evidence combined suggests SK proteins in axons, and not in dendrites, are important for nociception. As with all antibody staining approaches, it cannot be excluded that SK channels present in dendrites exist but are beneath the limits of detection by this approach (Walcott, 2018).

The finding on the axonal localization of SK channel proteins is interesting in several respects. First, it suggests that the enhanced Ca2+ signals that were observed in nociceptor soma are potentially caused by backpropagation of action potentials rather than a hyperexcitable soma or dendrites. Second, the proximal axonal localization is consistent with recently described evidence that other GFP-tagged Drosophila K+ channels (Shal and Elk) localize to the axon initial segment in the class I md neurons (Jegla, 2016). It is possible that SK regulates the AHP of action potentials as in other systems, and this, in turn, regulates firing frequency. It has been proposed that bursts and pauses in firing of the Drosophila nociceptor neurons may be necessary for robust nociception responses. Additionally, an 'unconventional spike' that is triggered by a large dendritic calcium transient has been proposed to be important. Indeed, a recent study also provided evidence that SK channels could regulate firing, because RNAi against SK was found to cause increases in the firing frequency of nociceptive neurons (Onodera, 2017). That study proposed that dendritically localized SK channels might respond to a dendritic Ca2+ transient. Although this investigation of the localization of the SK channel proteins makes them well positioned to regulate firing of nociceptive neurons, the finding that SK is localized to axons is inconsistent with the hypothesis that SK is directly regulated by dendritic Ca2+. Nevertheless, this comprehensive analysis, including the generation of null mutant alleles, and genomic and tissue-specific rescue experiments, demonstrates a genuine involvement for SK channels in Drosophila nociception. These studies of protein localization by a CRISPR engineered tagged SK channel, and anti-SK staining of an untagged rescuing cDNA suggest that the likely site of action for this important channel resides in the proximal axon segment and not in dendrites. An interesting question for the future will be to investigate whether Seizure and Tiwaz show a similar axonal localization (Walcott, 2018).

Synaptic excitation is regulated by the postsynaptic dSK channel at the Drosophila larval NMJ

In the mammalian CNS, the postsynaptic small-conductance Ca2+-dependent K+ (SK) channel has been shown to reduce postsynaptic depolarization and limit Ca2+ influx through NMDA receptors. To examine further the role of the postsynaptic SK channel in synaptic transmission, its action was studied at the Drosophila larval NMJ. Repetitive synaptic stimulation produced an increase in postsynaptic membrane conductance leading to depression of EPSP amplitude and hyperpolarization of the resting membrane potential (RMP). This reduction in synaptic excitation was due to the postsynaptic Drosophila SK (dSK) channel; synaptic depression, increased membrane conductance and RMP hyperpolarization were reduced in dSK mutants or after expressing a Ca2+ buffer in the muscle. Ca2+ entering at the postsynaptic membrane was sufficient to activate dSK channels based upon studies in which the muscle membrane was voltage clamped to prevent opening voltage-dependent Ca2+ channels. Increasing external Ca2+ produced an increase in resting membrane conductance and RMP that was not seen in dSK mutants or after adding the glutamate-receptor blocker philanthotoxin. Thus, it appeared that dSK channels were also activated by spontaneous transmitter release and played a role in setting membrane conductance and RMP. In mammals, dephosphorylation by protein phosphatase 2A (PP2A) increased the Ca2+ sensitivity of the SK channel; PP2A appeared to increase the sensitivity of the dSK channel since PP2A inhibitors reduced activation of the dSK channel by evoked synaptic activity or increased external Ca2+. It is proposed that spontaneous and evoked transmitter release activate the postsynaptic dSK channel to limit synaptic excitation and stabilize synapses (Gertner, 2014).

Stimulation of the neuromuscular synapses at 20 Hz resulted in an increase in resting Gin (input conductance), and a decrease in synaptic excitation due to membrane hyperpolarization and a decrease in EPSP amplitude. Simulations predicted that the increase in resting Gin during stimulation accounted for about one-third of the depression of EPSP amplitude due to shunting of the synaptic current. Synaptic depression is generally held to be due to a reduction in transmitter release; thus the remaining decrease in EPSP amplitude may have resulted from a decline in transmitter release. Nonetheless, evidence is provided for a change in postsynaptic conductance contributing to synaptic depression. The increase in Gin and RMP was consistent with activation of a gKCa; this was further supported by the reduced increase in Gin and RMP seen after expressing th Ca2+ buffer parvalbumin in the muscle (Gertner, 2014).

Larval muscle fibers contain two types of Ca2+-dependent K+ channels producing gCF and gCS. gCF was found to result from the slowpoke channel, a BK-type channel showing voltage- and Ca2+-dependent activation and voltage-dependent inactivation, and the dSK channel was proposed to produce gCS (Abou Tayoun, 2011). Western blots showed a single dSK isoform in larval brain and muscle; a Western blot of adult fly brains showed two closely spaced bands, which could have represented two dSK isoforms or a single isoform along with its posttranslationally altered form (Abou Tayoun, 2011). SK channels are not voltage-gated, and calmodulin acts as their Ca2+ sensor, resulting in a relatively high Ca2+ sensitivity. Repetitive synaptic stimulation activated the postsynaptic dSK channels, since the increase in Gin and RMP was eliminated in dSK or 24B/dSKDN larvae, but not in slo1 larva. As expected, there was reduced synaptic depression in dSK mutants and PV-expressing larvae; however, it was surprising that synaptic depression was completely eliminated. This suggests that there was also less depression of transmitter release in these larvae (Gertner, 2014).

It is assumed that the dSK channel is activated during evoked transmitter release in vivo, since the 20-Hz stimulation applied in this study likely falls within the physiological range of activity. The Ib synapses on muscle fiber 6 fire on average 20–30 Hz, and the Is terminals fire less than 10 Hz in dissected larvae; however, these firing frequencies may be greater in vivo since the waves of contraction are slow in dissected larvae, probably due to lack of appropriate sensory input. Also, since HL3 with low Ca2+ was used for the stimulation procedure, the postsynaptic Ca2+ influx was less than would occur under physiological conditions (Gertner, 2014).

Ca2+ entering at the postsynaptic membrane was sufficient to activate dSK channels, since evoked transmitter release produced an increase in Gin even when the muscle was voltage clamped at −60 mV. Under these conditions, it is expected that Ca2+ entry is limited to glutamate receptors, since voltage-dependent Ca2+ channels in larval muscle open at membrane potentials more positive than −30 mV. However, it cannot be ruled that there was a voltage drop across the subsynaptic reticulum (SSR), such that voltage-dependent Ca2+ channels near the glutamate receptors remained unclamped. In this case, both the glutamate receptors and nearby Ca2+ channels could have been the source of Ca2+ that activated the dSK channel. In fact, dSK channels in larval muscle can be activated by Ca2+ entering through voltage-dependent Ca2+ channels since depolarization of the muscle in the absence of synaptic activity produced an increase in gCS. Additional evidence that the glutamate receptor can act as the Ca2+ source comes from the activation of the dSK channel by spontaneous transmitter release; here, it seems unlikely that the SSR resistance would be great enough for quantal currents to open voltage-dependent Ca2+ channels. It appears that dSK channels exist at the postsynaptic membrane near the glutamate receptors, and this is consistent with findings in mammals, where the SK channel has been shown to be closely coupled to postsynaptic nicotinic acetylcholine receptors and NMDA receptors (Gertner, 2014).

The dSK channels were activated at rest and were responsible for the previously reported dependence of the RMP on external Ca2+. This was based upon the finding that the increase in Gin and RMP typically seen when increasing external Ca2+ from 0 to 1.5 mM was not seen in dSK larvae. In fact, it appeared that activation of the dSK channel is responsible for much of the normal variability in the RMP, since resting Gin and the RMP were positively correlated in wild-type larvae, but not in dSK larvae. Spontaneous transmitter release apparently activates dSK channels to set the resting Gin and RMP, since blocking the glutamate receptors prevented the rise in Gin and RMP produced by increasing external Ca2+. This is a novel function for spontaneous transmitter release, but not entirely unexpected, since spontaneous transmitter release produced postsynaptic Ca2+ transients similar in amplitude to evoked release (Gertner, 2014).

The spontaneous Ca2+ transients must have produced an increase in resting Gin that outlasted the Ca2+ signal, since the frequency of spontaneous events in this muscle is usually about 1 Hz, and the spontaneous Ca2+ transients had a decay time constant of ∼50 ms. The increase in Gin produced by repetitive synaptic stimulation also appeared to outlast the increase in [Ca2+]i, since it persisted for about 10 min after stimulation. Previous studies have found that after 5 s of 10-Hz stimulation, postsynaptic [Ca2+]i decayed with a time constant of about 100 ms, and this decay was largely due to the plasma membrane Ca2+ ATPase, which appeared to be highly enriched in the SSR. The SSR may limit Ca2+ diffusion to enhance the amplitude of postsynaptic Ca2+ transients; however, it seems likely that it also reduces their duration by providing efficient postsynaptic Ca2+ extrusion. Thus it seems improbable that postsynaptic [Ca2+]i remained elevated for 10 min after the stimulation train (Gertner, 2014).

There was an increase in leakage conductance (gL) in dSK larvae. This is consistent with previous studies showing covariation of ion conductances. In particular, reduced activation of Ca2+-dependent K+ currents in Drosophila cultured neurons resulted in a compensatory upregulation of the transient K+ current, apparently due to increased expression of transient K+ current channels. Although the channels responsible for gL have not been characterized in larval muscle, they may include ORK1 which was identified as a K+ leak channel in Drosophila and appears to be expressed in adult muscle; it is a member of the two-pore domain K+ channels identified in mammals. The nonselective cation channel NALCN produces the Na+ leak conductance in mammalian neurons and its ortholog na is expressed in Drosophila neurons, raising the possibility that it could also be responsible for the Na+ leak conductance in larval muscle (Gertner, 2014).

In mammals, CK2 and PP2A are constitutively bound to the SK channel and regulate the phosphorylation state of calmodulin, which determines the Ca2+ sensitivity and deactivation rate of the SK channel. Dephosphorylation of calmodulin by PP2A can reduce EC50 to 0.3 μM Ca2+ and phosphorylation of the SK channel by CK2 can increase EC50 to 2.0 μM. An increase in [Ca2+]i should shift the balance toward dephosphorylation and produce an increase in Ca2+ sensitivity, since CK2 cannot phosphorylate calmodulin when the channel is open. The current results showed that the dSK channel is also regulated by PP2A, since inhibiting PP2A reduced the increase in Gin and RMP produced by repetitive stimulation or increasing external Ca2+. During repetitive stimulation, the increase in postsynaptic [Ca2+]i may have increased the Ca2+ sensitivity of the dSK channel. Similarly, it may be that the Ca2+ transients produced by spontaneous transmitter release produced an increase in Ca2+ sensitivity so that the dSK channel is partially activated by resting [Ca2+]i. If the Ca2+-dependent increase in Ca2+ sensitivity persisted, this could explain the lasting increase in Gin seen after the end of 20-Hz stimulation and after spontaneous Ca2+ transients (Gertner, 2014).

The current results demonstrate that Ca2+ entering at the postsynaptic membrane during transmitter release provides negative feedback on synaptic excitation. This could rapidly stabilize the synapse and dampen the effects of changes in impulse activity, transmitter release or postsynaptic sensitivity. The effect of gSK on synaptic excitation was modeled using a Gsyn of 500 nS. The resting gSK in 1 mM (HL3) and 1.5 mM external Ca2+ (HL3.1) would make the EPSP peak about 3 mV and 14 mV more negative, respectively, mainly due to hyperpolarization of the RMP. An increase in gSK from 0 to 1 μS (approximately the value seen after 20 Hz stimulation) made the EPSP peak 22 mV more negative due to both RMP hyperpolarization and a decrease in EPSP amplitude. Activation of the dSK channel by evoked transmitter release is consistent with this negative-feedback mechanism, but what is the function of dSK channel activation by spontaneous transmitter release? minEPSP frequency is elevated after repetitive synaptic activity, and this could produce a persistent reduction in synaptic excitation. Also, since greater minEPSP frequency and amplitude would predict a larger EPSP, spontaneous transmitter release could set gSK to oppose strong synaptic excitation (Gertner, 2014).

This action of the postsynaptic dSK channel is similar to that found at mammalian central nervous system synapses, where the SK channel counteracts synaptic changes: experiments blocking SK channels with apamin, or overexpressing them, showed that activation of SK channels increased the threshold for the induction of LTP and impaired learning. At these synapses, it was proposed that SK channels act specifically to restrict Ca2+ entry through NMDA receptors limiting LTP and learning and memory. The current findings show that dSK channels play a more general role in regulating synaptic excitation (Gertner, 2014).

The dSK channel could act in concert with forms of homeostatic synaptic plasticity; these have involved compensatory changes in transmitter release at the NMJ, and most operate over long time scales. A failure of the dSK channel to maintain appropriate synaptic excitation could result in compensatory changes in transmitter release; this is supported by the apparent decrease in transmitter release seen when dSK channel activity was reduced in dSK mutants or by expressing PV in the muscle. This is consistent with previous findings that expressing additional K+ channels in Drosophila larval muscle resulted in a homeostatic increase in transmitter release (Gertner, 2014).

At mammalian central synapses, the SK channel reduced the amplitude of the EPSC; single EPSPs and EPSCs were larger after adding the SK-channel blocker apamin. Thus the SK channel was rapidly activated during synaptic transmission so that the resultant EPSC was a composite of the inward current through the postsynaptic receptor and outward current through the SK channel. This study did not directly examined whether the dSK channel influences the amplitude of the EPSC; this is made difficult since apamin does not block dSK channels. Evidence is available that dSK currents reduced the duration of the EPSC, but further experiments are required to characterize fully the contribution of the dSK current to the EPSC (Gertner, 2014).

Roles of the Drosophila SK channel (dSK) in courtship memory

A role for SK channels in synaptic plasticity has been very well-characterized. However, in the absence of simple genetic animal models, their role in behavioral memory remains elusive. This study took advantage of Drosophila melanogaster with its single SK gene (dSK) and well-established courtship memory assay to investigate the contribution of this channel to memory. Using two independent dSK alleles, a null mutation and a dominant negative subunit, this study showed that while dSK negatively regulates the acquisition of short-term memory 30 min after a short training session, it is required for normal long-term memory 24 h after extended training. These findings highlight important functions for dSK in courtship memory and suggest that SK channels can mediate multiple forms of behavioral plasticity (Abou Tayoun, 2012).

The Drosophila SK channel (dSK) contributes to photoreceptor performance by mediating sensitivity control at the first visual network

The contribution of the SK (small-conductance calcium-activated potassium) channel to neuronal functions in complex circuits underlying sensory processing and behavior is largely unknown in the absence of suitable animal models. This study generated a Drosophila line that lacks the single highly conserved SK gene in its genome (dSK). In R1-R6 photoreceptors, dSK encodes a slow Ca2+-activated K(+) current similar to its mammalian counterparts. Compared with wild-type, dSK(-) photoreceptors and interneurons showed accelerated oscillatory responses and adaptation. These enhanced kinetics were accompanied with more depolarized dSK(-) photoreceptors axons, assigning a role for dSK in network gain control during light-to-dark transitions. However, compensatory network adaptation, through increasing activity between synaptic neighbors, overcame many detriments of missing dSK current enabling dSK(-) photoreceptors to maintain normal information transfer rates to naturalistic stimuli. While demonstrating important functional roles for dSK channel in the visual circuitry, these results also clarify how homeostatically balanced network functions can compensate missing or faulty ion channels (Abou Tayoun, 2011).

This study provides the first description of an SK animal genetic model. Unlike mammals and C. elegans, which have three and four SK genes, respectively, the fruit fly genome contains a single highly conserved SK gene (dSK) that encodes a remarkably similar current to the mammalian counterpart. This report is also the first to evaluate the impact of SK channel on neuronal and network functions, which affect adaptation and signaling performance of photoreceptors. Because of this new model system, and the new insight it has given about sophisticated network functions, it is now possible to start dissecting the contributions of dSK channel and dSK-expressing cells in circuits, involved in complex behaviors, addiction, and learning and memory (Abou Tayoun, 2011).

The photoreceptor output is sign-inverted by large monopolar cells/amacrine cells' (LMCs/ACs') histamine-receptors and then partially fed back to photoreceptors through synaptic conductances (see dSK contributes to photoreceptor performance by fine-tuning synaptic transmission at the photoreceptor-LMC-photoreceptor network). Darkening hyperpolarizes photoreceptors, reducing their tonic histamine release. This in turn depolarizes LMCs/ACs, increasing their feedbacks to photoreceptor axons. These excitatory conductances can explain why in the dark, photoreceptors of the fully functioning network are more depolarized than the dissociated photoreceptors, which lack axons (Abou Tayoun, 2011).

dSK contributes to photoreceptor performance by fine-tuning synaptic transmission at the photoreceptor-LMC-photoreceptor network. In this model, dSK is expressed in photoreceptor axons, while glutamatergic AC interneurons and L2 monopolar cells, which form the direct synaptic feedbacks to photoreceptor axons, do not express dSK. In addition, photoreceptor axons and AC/L2 receive extra inputs from the lamina network, through functional contacts with nonglutamatergic LMCs that express dSK, including L4s. Within R1-R6 axons, dSK counteracts Ca2+ and fine-tunes neurotransmitter release. In the dark, LMCs receive less feedforward input from photoreceptors and are, therefore, more depolarized. In the absence of dSK, the observed depolarized resting potential and the lowered input resistance can be attributed to an inability to zero photoreceptor axonal voltages and/or increased feedback synaptic inputs into photoreceptor axons due to misregulated histamine release. In the light, photoreceptors are more depolarized and, therefore, the feedback input is reduced (both direct and extra). In the dark/dim conditions, an inability to fine-tune neurotransmitter release from nonglutamatergic LMCs (most likely L4), at least partially, can lead to the observed oscillatory responses in both photoreceptors and LMCs (particularly in L2s) in dSK- flies (Abou Tayoun, 2011).

At the photoreceptor terminals, the slowly hyperpolarizing K+ conductances of dSK channels are likely to facilitate local inhibition by offsetting voltage-dependent Ca2+ increases, as a part of axonal sensitivity control that refines waveforms and patterning of presynaptic signals. Here, dSK might be also required in fine-tuning histamine release, whereby any synaptic transmission defect would lead to increased feedback inputs into photoreceptor axons. These scenarios are not mutually exclusive and in the absence of dSK would result in increased synaptic feedback conductances from the lamina interneurons, leading to the observed lowered input resistance and the more depolarized resting potential of photoreceptors. They are further supported by the expression data, and the intact morphology and phototransduction machinery in dSK- photoreceptors (Abou Tayoun, 2011).

During and following prolonged light exposure, both dSK- photoreceptor and LMC voltage responses oscillated in an activity-dependent manner, implying that in wild-type, dSK channels would have a direct role as adaptive dampers in synaptic communication. It is possible that these perturbations resulted from faulty synaptic gain control in dSK-expressing nonglutamatergic interneurons, because inactivation of dSK only in photoreceptors was not sufficient to induce them, and because in mutant, dSK seemed not expressed in glutamatergic interneurons (ACs and L2s), which synapse directly to photoreceptor axons. Furthermore, photoreceptor oscillations were strengthened after light adaptation and to dim but not bright luminances. Such stimulus conditions lower presynaptic (photoreceptor) potentials and, therefore, are expected to boost postsynaptic feedbacks from the network. Thus, in dim luminances, mistuned synaptic feedbacks of high gain could transfer energy to wrong stimulus frequencies, oscillating the photoreceptor output. Collectively, these findings hint that oscillations might originate from dSK- L4 monopolar cell synapses, which feedback to both photoreceptor axons and L2 monopolar cells in the same and neighboring lamina cartridges, and thus are ideally placed to mediate adaptive network functions (Abou Tayoun, 2011).

To maximize visual information, network adaptation to dim environment involves integration over space and time, whereupon functional connectivity increases between cells (redundancy), smoothing low signal-to-noise images. But when adapting to bright environment its cells operate more independently, as lateral and temporal inhibition reduces redundancies to sharpen high signal-to-noise images. Thus, it is speculated that faulty gain control in the lamina branches of dSK- L4 monopolar cells would hinder such connectivity transitions between dark- and light-adapted network states; affecting the rate of adaptation in photoreceptor and LMC outputs and making them more susceptible to oscillations. This view is further supported by the recording statistics from dSK- flies: all R1-R6 receive inputs from L4, and correspondingly most photoreceptor outputs showed suboptimal adaptation and oscillated; only L2 monopolar cells receive inputs from L4 and only ~40% of LMC outputs oscillated. These observations highlight how the neuronal functions in the early motion pathways can depend upon adaptive gain control, leading to different behavioral outcomes in different stimulus conditions (Abou Tayoun, 2011).

Despite the fast oscillatory responses, dSK- photoreceptors revealed a near-normal encoding capacity. The decreased input resistance in dSK- photoreceptors is similar to that found in Shaker and Shab mutant photoreceptors, where it has been argued to compensate for mutant defects and underlie the robustness of encoding. In Shaker photoreceptors, the decrease in input resistance partially restores the efficient use of the operating voltage range. Conversely, dSK- photoreceptors, like Shab, show remarkable robustness in their light-voltage relationships, sensitivities, and reliability of dynamic encoding (Abou Tayoun, 2011).

Although it is unclear whether the underlying mechanisms are the same, the lower input resistances in all these mutant photoreceptors are believed to be direct manifestations of compensation. In this study, the lowered input resistance, measured from intact dSK- photoreceptors in vivo, indicates an increase in conductance at the photoreceptor axon. This study proposes that this compensation results from feedback synaptic inputs from the neighboring interneurons, because everything else being equal, instead of depolarizing dSK- photoreceptors, excess of K+ and/or Cl- leak-conductances would work to hyperpolarize the cells toward the reverse potentials of these ions (-80 mV). Thus, even if such leaks existed - a possibility that cannot be excluded - they would be masked by the depolarizing synaptic conductances from the network. Furthermore, response dynamics of photoreceptors, with inactivated dSK channels, were closer to wild-type when the network was normal rather than mutated, implying that extrinsic conductances (from the network) shape photoreceptor output more than intrinsic leak conductances (which, if dominating, should produce identical outputs for the two cases) (Abou Tayoun, 2011).

The faster kinetics and retuned adapting properties of dSK- photoreceptors impose a constant high energy cost to maintain both a low input resistance and a depolarized resting potential in the dark, suggesting that the mutants are at a clear disadvantage. Thus, managing energy costs is a powerful evolutionary objective, which together with noise and various behavioral objectives, supposedly refined the molecular constituents of the lamina network to overcome the limitations of its unreliable, slow hardware (Abou Tayoun, 2011 and references therein).

Functions of SK channel orthologs in other species

A V-to-F substitution in SK2 channels causes Ca(2+) hypersensitivity and improves locomotion in a C. elegans ALS model

Small-conductance Ca(2+)-activated K(+) (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca(2+)-bound calmodulin. An intrinsically disordered fragment has been identified that is essential for the mechanical coupling between Ca(2+)/calmodulin binding and channel opening. This study reports that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca(2+) sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP2-interacting residue K405, and subsequently enhanced Ca(2+) sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca(2+) hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca(2+) with increased activity in vivo (Nam, 2018).

Activity-dependent plasticity of spike pauses in cerebellar purkinje dells

The plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst-pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, this study found that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2(-/-) mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. These findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity-by altering the Purkinje cell output-may be crucial to cerebellar information storage and learning (Grasselli, 2016).

Structural insights into the potency of SK channel positive modulators

Small-conductance Ca(2+)-activated K(+) (SK) channels play essential roles in the regulation of cellular excitability and have been implicated in neurological and cardiovascular diseases through both animal model studies and human genetic association studies. Over the past two decades, positive modulators of SK channels such as NS309 and 1-EBIO have been developed. Previous structural studies have identified the binding pocket of 1-EBIO and NS309 that is located at the interface between the channel and calmodulin. This study took advantage of four compounds with potencies varying over three orders of magnitude, including 1-EBIO, NS309, SKS-11 (6-bromo-5-methyl-1H-indole-2,3-dione-3-oxime) and SKS-14 (7-fluoro-3-(hydroxyimino)indolin-2-one). A combination of x-ray crystallographic, computational and electrophysiological approaches was utilized to investigate the interactions between the positive modulators and their binding pocket. A strong trend exists between the interaction energy of the compounds within their binding site calculated from the crystal structures, and the potency of these compounds in potentiating the SK2 channel current determined by electrophysiological recordings. These results further reveal that the difference in potency of the positive modulators in potentiating SK2 channel activity may be attributed primarily to specific electrostatic interactions between the modulators and their binding pocket (Nam, 2017).

SK channel inhibition mediates the initiation and amplitude modulation of synchronized burst firing in the spinal cord

Burst firing in motoneurons represents the basis for generating meaningful movements. Neuromodulators and inhibitory receptor blocker cocktails have been used for years to induce burst firing in vitro; however, the ionic mechanisms in the motoneuron membrane that contribute to burst initiation and amplitude modulation are not fully understood. Small conductance Ca(2+)-activated potassium (SK) channels regulate excitatory inputs and firing output of motoneurons and interneurons and therefore, are a candidate for mediating bursting behavior. The present study examines the role of SK channels in the generation of synchronized bursting using an in vitro spinal cord preparation from adult mice. These results show that SK channel inhibition is required for both initiation and amplitude modulation of burst firing. Specifically, administration of the synaptic inhibition blockers strychnine and picrotoxin amplified the spinal circuit excitatory drive but not enough to evoke bursting. However, when SK channels were inhibited using various approaches, the excitatory drive was further amplified, and synchronized bursting was always evoked. Furthermore, graded SK channel inhibition modulated the amplitude of the burst in a dose-dependent manner, which was reversed using SK channel activators. Importantly, modulation of neuronal excitability using multiple approaches failed to mimic the effects of SK modulators, suggesting a specific role for SK channel inhibition in generating bursting. Both NMDA (N-methyl-d-aspartate) and AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptors were found to drive the synchronized bursts. The blocking of gap junctions did not disturb the burst synchrony. These results demonstrate a novel mechanistic role for SK channels in initiating and modulating burst firing of spinal motoneurons (Mahrous, 2017).


Search PubMed for articles about Drosophila SK

Abou Tayoun, A. N., Li, X., Chu, B., Hardie, R. C., Juusola, M. and Dolph, P. J. (2011). The Drosophila SK channel (dSK) contributes to photoreceptor performance by mediating sensitivity control at the first visual network. J Neurosci 31(39): 13897-13910. PubMed ID: 21957252

Abou Tayoun, A. N., Pikielny, C. and Dolph, P. J. (2012). Roles of the Drosophila SK channel (dSK) in courtship memory. PLoS One 7(4): e34665. PubMed ID: 22509342

Gertner, D. M., Desai, S. and Lnenicka, G. A. (2014). Synaptic excitation is regulated by the postsynaptic dSK channel at the Drosophila larval NMJ. J Neurophysiol. PubMed ID: 24671529

Grasselli, G., He, Q., Wan, V., Adelman, J. P., Ohtsuki, G. and Hansel, C. (2016). Activity-Dependent Plasticity of Spike Pauses in Cerebellar Purkinje Cells. Cell Rep 14(11): 2546-2553. PubMed ID: 26972012

Hu, C., Petersen, M., Hoyer, N., Spitzweck, B., Tenedini, F., Wang, D., Gruschka, A., Burchardt, L. S., Szpotowicz, E., Schweizer, M., Guntur, A. R., Yang, C. H. and Soba, P. (2017). Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior. Nat Neurosci 20(8): 1085-1095. PubMed ID: 28604684

Hwang, R. Y., Zhong, L., Xu, Y., Johnson, T., Zhang, F., Deisseroth, K. and Tracey, W. D. (2007). Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr Biol 17(24): 2105-2116. PubMed ID: 18060782

Jegla, T., Nguyen, M. M., Feng, C., Goetschius, D. J., Luna, E., van Rossum, D. B., Kamel, B., Pisupati, A., Milner, E. S. and Rolls, M. M. (2016). Bilaterian giant ankyrins have a common evolutionary origin and play a conserved role in patterning the axon initial segment. PLoS Genet 12(12): e1006457. PubMed ID: 27911898

Mahrous, A. A. and Elbasiouny, S. M. (2017). SK channel inhibition mediates the initiation and amplitude modulation of synchronized burst firing in the spinal cord. J Neurophysiol 118(1): 161-175. PubMed ID: 28356481

Nam, Y. W., Orfali, R., Liu, T., Yu, K., Cui, M., Wulff, H. and Zhang, M. (2017). Structural insights into the potency of SK channel positive modulators. Sci Rep 7(1): 17178. PubMed ID: 29214998

Nam, Y. W., Baskoylu, S. N., Gazgalis, D., Orfali, R., Cui, M., Hart, A. C. and Zhang, M. (2018). A V-to-F substitution in SK2 channels causes Ca(2+) hypersensitivity and improves locomotion in a C. elegans ALS model. Sci Rep 8(1): 10749. PubMed ID: 30013223

Onodera, K., Baba, S., Murakami, A., Uemura, T. and Usui, T. (2017). Small conductance Ca(2+)-activated K(+) channels induce the firing pause periods during the activation of Drosophila nociceptive neurons. Elife 6. PubMed ID: 29035200

Terada, S., Matsubara, D., Onodera, K., Matsuzaki, M., Uemura, T. and Usui, T. (2016). Neuronal processing of noxious thermal stimuli mediated by dendritic Ca(2+) influx in Drosophila somatosensory neurons. Elife 5. PubMed ID: 26880554

Walcott, K. C. E., Mauthner, S. E., Tsubouchi, A., Robertson, J. and Tracey, W. D. (2018). The Drosophila small conductance calcium-activated potassium channel negatively regulates nociception. Cell Rep 24(12): 3125-3132. PubMed ID: 30231996

Biological Overview

date revised: 17 November 2021

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.