Scott, Luisa et al. (2013) International Worm Meeting "Identifying Novel BK Channel Modulators."

Scott, Luisa, Davis, Scott, Shen, Angela, Nordquist, Sarah, Philpo, Ashley, Iyer, Sangeetha, Pierce-Shimomura, Jon

[International Worm Meeting, 2013]

The large-conductance calcium-activated potassium (BK) channel is an evolutionarily conserved target of ethanol. Physiologically relevant concentrations of ethanol increase BK channel opening. Additionally, genetic manipulation of BK channel function affects behavioral sensitivity to ethanol in multiple animal models. These findings suggest that the BK channel is a viable therapeutic target for alcohol intoxication and addiction. To begin to test this, we have developed a screen to identify peptides that alter BK channel function. First, we screened 30 million 9 amino acid peptide sequences using a monovalent phagemid display technique. Twenty-seven unique peptides remained after panning for sequences that bind to the human BKa channel but not the rat SK2 channel or human glycine a1 receptor. Sequences with clusters of positively charged amino acids were enriched 100-300 fold. Three motifs were enriched 3000-6000 fold. Next, select peptides were rapidly screened for functional effects using the nematode, C. elegans. Several peptides with highly enriched motifs affected an ethanol- and BK-channel dependent behavior in this animal. The behavioral assay suggests that peptide pskan4 enhances the ethanol mediated increase in BK channel opening, but does not alter channel activity in the absence of ethanol. Peptide pskan1 had BK channel- but not ethanol-dependent effects. In contrast, a peptide without a highly enriched motif had non-specific effects, and a 9 amino acid peptide not selected in the panning procedure did not alter locomotion. Finally, we have begun to confirm the function of the peptides electrophysiologically. Preliminary recordings support the putative action of peptides pskan1 and pskan4 as BK channel openers. Overall, these findings show that we have developed and successfully employed a screen for identifying and characterizing novel peptides that alter BK channel-dependent behavior in the presence of physiologically relevant concentrations of ethanol. This screening technique can be applied to identify modulators of other ion channels.

Davis S J et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Uncovering the Molecular Basis for Ethanol Activation of the BK Channel Using a Genetic Screen"

Davis S J, Song S S, Pierce-Shimomura J T

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Ethanol acts on dozens of ion channel types across many species. However, the molecular basis for how ethanol affects these ion channels is poorly understood, as are their individual contributions to altered behavior. Previously, a genetic screen in C. elegans defined the voltage and calcium-activated BK potassium channel as the major target of ethanol to produce behavioral intoxication (Davies et al., 2003). Moreover, the BK channel was activated by typical human intoxication conditions of 20 mM ethanol with in vivo patch-clamp recording. The BK channel has recently been implicated in sensitivity to intoxication in humans, and tolerance in both mice and Drosophila. The genetic tractability of C. elegans allows for rapid screening of mutations in the BK channel gene. In vitro studies have already identified specific regions and residues of the BK channel that are critical for BK channel activation by ethanol. We hypothesize that amino acid residues in the BK channel exist that when mutated render the channel insensitive to activation by ethanol in vivo, without severely compromising channel function. Such mutations might reveal potential binding sites for ethanol or parts of the channel that undergo ethanol-induced conformational changes. To test this hypothesis, we are performing a large-scale screen to obtain novel mutations in the BK channel that confer behavioral resistance to ethanol in vivo. Currently, this ongoing screen has uncovered 20 candidate BK channel mutants that demonstrate resistance to intoxication. In order to predict whether new mutants are null or non-null we are quantitatively comparing the pattern of movement made by these mutants compared to those made by wild type and a known null mutant. Non-null candidate mutants will undergo genetic sequencing and further analyzed with in vivo patch-clamp recordings to reveal how mutations change the basal properties and/or ethanol sensitivity of the BK channel. Uncovering residues critical to behavioral intoxication will elucidate the molecular interaction between ethanol and the BK channel that can be tested further in mammalian species.

Davis, Scott et al. (2013) International Worm Meeting "Uncovering the molecular basis for ethanol action on the BK channel using genetic screens."

Hu, Kevin, Pierce-Shimomura, Jon, Davis, Scott

[International Worm Meeting, 2013]

Development of alcohol abuse has a strong genetic component. People who are innately resistant to intoxication are at high risk to abuse alcohol and vice versa. This genetic predisposition is linked to variation in genes that encode targets of alcohol or their downstream effectors involved in intoxication. Genetic screens in C. elegans previously determined that the gene slo-1 represents a central target for alcohol intoxication in the worm. slo-1 encodes the highly conserved large-conductance, calcium-activated potassium (BK) channel. The BK channel has emerged as a key mediator for intoxication and tolerance across species from worm, fly, mouse to human. At the physiological level, BK channel activity is modulated by clinically relevant concentrations of ethanol (~20mM). To reveal specific residues in the BK channel that are required for activation by alcohol, and thus may influence sensitivity to behavioral intoxication, we are performing multiple genetic screens. First, we are screening slo-1 mutants from the million mutation project. Second, we are performing site-directed mutagenesis on the worm and human BK channels. We are able to study the human BK channel in the worm because we rescued ethanol sensitivity in a slo-1 null worm by expression of hslo. Novel mutants that are resistant to intoxication will either contain 1) a null mutation in slo-1 that provides no new information, or 2) a novel non-null mutation that provides insight into how specific residues on the BK channel interact with alcohol, and how this interaction alters intoxication. To distinguish non-null candidates, the locomotor posture of mutants recovered from the genetic screens will be compared against wild-type and a known slo-1 null mutant. Candidate non-null mutants will display different locomotor posture than the known slo-1 null mutant. Currently, we have isolated multiple candidate non-null mutants with resistance to intoxication. Further analysis using in vivo patch-clamp recordings will assess how specific mutations alter basal BK channel function and the response to alcohol.

Chen, Bojun et al. (2009) International Worm Meeting "BKIP-1 is a Novel Regulatory Protein Critical to SLO-1 Function in vivo."

Ge, Qian, Zhan, Haiying, Wang, Zhao-Wen, Chen, Bojun

[International Worm Meeting, 2009]

The BK channel is a large-conductance potassium channel gated by both membrane potential and intracellular calcium. It is almost ubiquitously expressed and performs many important physiological functions, including shaping action potential wave form and regulating neurotransmitter release. The C. elegans BK channel SLO-1 is expressed in most, if not all, neurons and several types of muscles (body-wall, vulval, and anal depressor muscles). BK channel functional properties are tuned to specific cellular needs through a variety of mechanisms, including association with auxiliary/regulatory proteins. Several BK channel auxiliary/regulatory proteins have been identified in mammals (known as b1-, b2-, b3, and b4-subunits) and Drosophila (known as Slob and dSLIP1). However, none of them has obvious homologues in C. elegans. It is likely that there are other unidentified BK channel auxiliary/regulatory proteins. In an attempt to identify the putative additional BK channel auxiliary/regulatory proteins, we performed a genetic screen for mutants that suppressed the lethargic phenotype caused by expressing a slo-1 gain-of-function (gf) transgene. This analysis led to the identification of bkip-1 (BK channel interacting protein 1), which encodes a putative integrative membrane protein distinct from any of the known BK channel auxiliary/regulatory proteins. bkip-1 mutants completely or near completely suppressed the lethargic phenotype of slo-1(gf), and were grossly indistinguishable from slo-1 loss-of-function (lf) mutants in locomotion behaviors. Analyses of postsynaptic currents at the neuromuscular junction showed that neurotransmitter release was similarly increased in bkip-1(lf) mutant as in slo-1(lf) mutant, and that bkip-1(lf) eliminated an inhibitory effect of slo-1(gf) on neurotransmitter release. In bkip-1 mutants, slo-1 transcription was normal but SLO-1 protein level in the nerve ring was significantly reduced, suggesting that BKIP-1 may play a role in SLO-1 synthesis, surface expression or localization. The expression pattern of bkip-1 was grossly indistinguishable from that of slo-1. When analyzed in the Xenopus oocyte expression system, BKIP-1 increased SLO-1 membrane current density, and, in a Ca2+-dependent manner, slowed SLO-1 activation and shifted the conductance-voltage (G-V) relationship. These observations establish BKIP-1 as an important SLO-1 regulatory protein in C. elegans.

Davis, Scott J et al. (2011) International Worm Meeting "Uncovering the molecular basis for ethanol activation of the BK channel using random mutagenesis."

Pierce-Shimomura, Jon, Davis, Scott J, Milman, Kelly, Song, Sam

[International Worm Meeting, 2011]

Previous research has demonstrated a strong genetic component in the development of alcohol abuse. Specifically, people with a high resistance to intoxication at the time of their first drink are at risk. This suggests protein targets of alcohol or their downstream effectors that mediate behavioral intoxication are critical for the potential of an individual to abuse alcohol. A highly conserved target protein that is emerging as a key mediator for behavioral intoxication and tolerance is the big conductance potassium (BK) channel(1). The BK channel is activated by low levels of alcohol (~20 mM) across many species including C. elegans, rodent and human, which is equivalent to the legal level of intoxication. In mice, this channel contributes to both behavioral intoxication and tolerance. A gain-of function mutation in the BK channel results in hypersensitivity to alcohol in humans 2. In addition, a genetic screen identified a null mutation in the slo-1 gene, which encodes the BK channel, as critical for resistance to behavioral intoxication in C. elegans3. In order to elucidate the interaction of ethanol with the BK channel at the molecular level, we are using a genetic screen to uncover novel non-null mutations throughout the SLO-1 channel that result in resistance to behavioral intoxication. In addition, we are using random mutagenesis to uncover novel non-null mutations in the C-terminus of the SLO-1 channel, a region previously implicated in the response to ethanol in mice4. Non-null candidates will be distinguished from null mutants by differences in locomotory pattern and subsequent sequence analysis. So far, one of the 20 mutants has been identified as a candidate non-null mutant. After a non-null mutant is identified, we will perform in vivo patch-clamp recordings to assess how the mutation alters basal BK channel function and the response to alcohol. This study may provide knowledge on how ethanol acts on the BK channel at the molecular level to induce intoxication across species. 1. Treistman and Martin. BK Channels: mediators and models for alcohol tolerance, Trends Neurosci. 2009. 32(12)629-37; 2. Du et al., Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder Nat Genet. 2005. 733-8; 3. Davies et al., A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell. 2003. 115(6) 655-66; 4. Liu et al., Ethanol modulates BKCa channels by acting as an adjuvant of calcium. Mol Pharmacol. 2008. 74(3) 628-40.

Brandon E Johnson et al. (2005) International Worm Meeting "A genetic approach to locate Ca2+-sensitive sites on the slo-1 BK channel"

Richard W Aldrich, Miriam B Goodman, Brandon E Johnson

[International Worm Meeting, 2005]

How ligand binding opens ion channels is poorly understood. We are using the large-conductance, Ca2+-activated potassium (BK) channel to study this question. BK channels are activated by Ca2+ binding and by membrane depolarization. This dual regulation allows the Ca2+ activation pathway to be attenuated or eliminated, while maintaining a functional channel that is voltage-activated. Previous studies used site-directed approaches to locate regions of the BK channel necessary for Ca2+-dependent gating and for Ca2+ binding to an isolated C-terminal fragment. These directed approaches have led to conflicting results: mutations that alter conserved regions of the C-terminal tail dramatically decrease Ca2+ sensitivity, while truncated channels lacking this entire domain retain normal Ca2+ sensitivity. To locate additional regions of the BK channel important for Ca2+ sensitivity, we are developing a mutant screen in C. elegans. To do this, we are using aldicarb sensitivity as a behavioral readout of motor neuron function. slo-1 null mutants increase aldicarb sensitivity and duration of synaptic vesicle release at the neuromuscular junction. Conversely, unc-2 partial loss-of-function and null mutants reduce aldicarb sensitivity and, presumably, acetylcholine release. As a proof-of-principle for our screen, we tested the aldicarb sensitivity of unc-2;slo-1 double mutants. We found that the aldicarb sensitivity of unc-2(null);slo-1 was similar to unc-2(null), while that of unc-2(lf);slo-1 was similar to wild type. From these results, we infer that the Ca2+ concentration in unc-2(lf) motor neurons is sufficient to activate BK channels. We are using this genetic interaction as the basis for a screen for additional slo-1 alleles, looking for mutations that enhance aldicarb sensitivity in unc-2(lf) animals. We have recovered 25 aldicarb-sensitive mutants, including several that fail to complement unc-2(lf);slo-1. We plan to determine how these newly identified mutations affect BK channel function by measuring their Ca2+ and voltage dependence in Xenopus oocytes. These data will be used to construct a model of protein domains involved in BK channel activation.

Brandon Johnson et al. (2008) C.elegans Neuronal Development Meeting "The Ensemble of BK Channel Splice Variants in Caenorhabditis elegans"

Richard Aldrich, Brandon Johnson, Miriam Goodman

[C.elegans Neuronal Development Meeting, 2008]

Large-conductance, Ca- and voltage-activated potassium (BK) channels regulate many functions including neurotransmitter release at the neuromuscular junction (NMJ). The pore-forming subunit of BK channels is transcribed from a single gene, slo-1, which encodes multiple isoforms through alternative splicing. To better understand how splicing affects BK channel function, we sought to identify and characterize all slo-1 splice variants from a single organism. As the number of potential splice variants exceeds 1,000 for flies and mammals, we focused on C. elegans in which slo-1 contains three splice sites and only 12 possible splice variants. Using standard techniques in RT-PCR and restriction digestion, we developed a novel method to determine which of the possible combinations of alternative exons are expressed as unique splice variants. We identified all twelve predicted slo-1 splice variants; this set includes three previously reported variants and nine new ones. To determine how alternative splicing affects BK channel function, we are measuring the Ca- and voltage-dependence of each splice variant expressed in heterologous cells. These isoforms show a range of apparent Ca and voltage sensitivities, supporting the idea that alternative splicing is a mechanism to regulate BK channel activation in diverse cell types. We identified a slo-1 allele (pg34) that encodes a single amino acid substitution in an alternatively spliced exon, which affects eight of the 12 slo-1 splice variants. Intriguingly, we find that this mutation has a minor effect on channel function in one isoform, while significantly altering channel activity in another isoform. pg34 mutants are hypersensitive to paralysis induced by an acetylcholine esterase inhibitor (aldicarb), but show wild-type sensitivity to an acetylcholine receptor agonist, levamisole. Thus, pg34 is likely to act presynaptically at the NMJ. Together, these data suggest that pg34 reduces but does not eliminate channel activity in slo-1 splice variants needed for proper BK channel function at the neuromuscular junction.

Qiang Liu et al. (2007) International Worm Meeting "Presynaptic CaMKII Modulates Neurotransmitter Release by Activating BK Channels at C. elegans Neuromuscular Junction."

Bojun Chen, Qiang Liu, Qian Ge, Zhao-Wen Wang

[International Worm Meeting, 2007]

CaMKII plays key roles in controlling synaptic strength and plasticity. Although the function of postsynaptic CaMKII is well-known, that of presynaptic CaMKII is poorly defined. To understand the function of presynaptic CaMKII, it is important to identify its molecular targets. We assessed the function of presynaptic CaMKII in neurotransmitter release and the possibility of the BK channel as a mediator of presynaptic CaMKII function by analyzing miniature and evoked postsynaptic currents at the C. elegans neuromuscular junction. The BK channel was chosen as a candidate because (1) it plays a major role in regulating neurotransmitter release, (2) it may be activated by CaMKII, and (3) loss-of-function mutants of both unc-43 CaMKII and slo-1 BK channel were isolated in the same genetic screen as suppressors of the lethargic phenotype of a hypomorphic unc-64 syntaxin mutant (Wang et al., 2001). We found that either loss-of-function (lf) or gain-of-function (gf) of unc-43 inhibited neurotransmitter release. The inhibitory effect of unc-43(gf) was reversed by mutation or blockade of SLO-1. SLO-1 expressed in Xenopus oocytes could be activated by recombinant rat <font face=symbol>a</font>-CaMKII, and this effect of CaMKII was abolished by mutating a threonine residue (T425) at a consensus CaMKII phosphorylation site in the first RCK domain (regulator of conductance for K+) of the channel. The inhibitory effect of unc-43(gf) was not reversed by unc-103(lf), dgk-1(lf), or eat-16(lf), which may suppress behavioral phenotypes of unc-43(gf) (Robatzek and Thomas, 2000). These observations suggest that presynaptic CaMKII is a bidirectional modulator of neurotransmitter release, presumably by phosphorylating different molecular targets, and that its negative modulatory effect on the release is mainly mediated by SLO-1 activation.

Clites, Benjamin et al. (2021) International Worm Meeting "Genetic architecture of alcohol sensitivity in C. elegans"

Andersen, Erik, Pierce, Jonathan, Palacios, Bridgitte, Clites, Benjamin, Evans, Kathryn

[International Worm Meeting, 2021]

Developing medical treatments for alcohol use disorders (AUD) may benefit from identifying molecules that underlie behavioral responses to alcohol. Forward genetics and candidate gene screens using C. elegans have identified molecules required for behavioral responses to alcohol, including the BK potassium channel, which is a direct target of alcohol, as well as the CRF and SWI/SNF pathways, which modify alcohol responses. These and other molecules identified in C. elegans appear to work in conserved ways in mammalian models of AUD and humans. Despite their success, genetic screens likely miss other genes of large effect if, for example, those genes are essential for development, functionally redundant, or are rare. By leveraging natural phenotypic and genetic variation in 200 wild isolates from across the globe, rather than mutagenesis using the lab strain N2, we uncovered new conserved genes that contribute to alcohol sensitivity in C. elegans. Our genome-wide association study revealed four significant quantitative trait loci (QTLs), three of which have never been implicated in alcohol response in nematodes or mammals, as well as the BK channel gene, slo-1, which validated our approach. Candidate QTL genes included an FBOXA gene located within an expanded cluster of other FBOX genes, an essential nuclear transporter gene, and a rare isoform-specific allele of protein kinase D (dkf-2). Additionally, we found that deletion of dkf-2 in an N2 background conferred high resistance to intoxication. One extremely resistant wild isolate, JU830, which harbors all resistant genotypes at the QTLs, was six standard deviations above mean resistance. In contrast to the small indel and SNP variation associated with the four QTL in other strains, closer inspection of the BK channel slo-1 locus in JU830 identified large indels in a number of its introns as well as its 5'UTR. Our results suggest that C. elegans underwent selection for alcohol sensitivity and resistance in nature. The three novel genes identified here, especially the protein kinase D, might represent druggable targets for novel treatments for alcohol addiction and abuse.

Jon Pierce-Shimomura et al. (2002) Japanese Worm Meeting "Behavioral responses to ethanol are mediated by a BK potassium channel in C. elegans"

Antonello Bonci, Steve McIntire, Hongkyun Kim, Jon Pierce-Shimomura, Tod R Thiele, Andrew Davies

[Japanese Worm Meeting, 2002]

We screened for mutant C. elegans that are resistant to intoxication by ethanol. We found that strong resistance is conferred by mutations in the gene slo-1 , which encodes a highly conserved ortholog of the human large conductance calcium-activated potassium (BK) channel 1-3 . Prevous work has shown effects of ethanol on vertebrate BK channels in vitro 4-9 . We hypothesized that intoxication in C. elegans may be primarily mediated by ethanol activating the SLO-1 channel; thus strong resistance to intoxication by ethanol results when the SLO-1 channel is absent. To test this idea, we first performed in vivo whole-cell voltage-clamp recordings of dopamine neurons to identify a SLO-1 current that was present in neurons in wild-type worms but absent in slo-1 mutants. Next, we found that ethanol selectively and reversibly increased the amplitude of the outward-rectifying current in neurons of wild-type animals at physiologically relevant concentrations (20 & 100 mM). Ethanol had no effect, however, on currents in neurons of slo-1 mutants. To determine whether ethanol can activate SLO-1 directly in the absence of cytosolic factors, we recorded the single-channel activity of SLO-1 in patches excised from dopamine neurons. Ethanol reversibly increased the probability of channel opening. Thus, we have demonstrated a direct relationship between the SLO-1 channel, a biochemical effect of ethanol and a genetic effect on behavior 1 Wei et al (1996) Neuropharm; 2 Wang et al (2001) Neuron; 3 Chu et al (1998) Mol Pharmacol; 4 Dopico et al (1998) J Pharmacol Exp Ther; 5 Dopico et al (1996) Mol Pharmacol; 6 Dopico et al (1999) J Physiol; 7 Gruss et al (2001) Eur J Neurosci; 8 Jakab et al (1997) J Membr Biol; 9 Walters et al (2000) Am J Physiol Cell Physiol