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Opioid-induced respiratory depression (OIRD) represents the primary cause of death associated with therapeutic and recreational opioid use. Within the United States, the rate of death from opioid abuse since the early 1990s has grown disproportionally, prompting the classification as a nationwide “epidemic.” Since this time, we have begun to unravel many fundamental cellular and systems-level mechanisms associated with opioid-related death. However, factors such as individual vulnerability, neuromodulatory compensation, and redundancy of opioid effects across central and peripheral nervous systems have created a barrier to a concise, integrative view of OIRD. Within this review, we bring together multiple perspectives in the field of OIRD to create an overarching viewpoint of what we know, and where we view this essential topic of research going forward into the future.

Because hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels modulate the excitability of cortical and hippocampal principal neurons, these channels play a key role in the hyperexcitability that occurs during the development of epilepsy after a brain insult, or epileptogenesis. In epileptic rats generated by pilocarpine-induced status epilepticus, HCN channel activity is downregulated by two main mechanisms: a hyperpolarizing shift in gating and a decrease in amplitude of the current mediated by HCN channels, I(h). Because these mechanisms are modulated by various phosphorylation signaling pathways, we hypothesized that phosphorylation changes occur at individual HCN channel amino acid residues (phosphosites) during epileptogenesis. We collected CA1 hippocampal tissue from male Sprague Dawley rats made epileptic by pilocarpine-induced status epilepticus, and age-matched naïve controls. We also included resected human brain tissue containing epileptogenic zones (EZs) where seizures arise for comparison to our chronically epileptic rats. After enrichment for HCN1 and HCN2 isoforms by immunoprecipitation and trypsin in-gel digestion, the samples were analyzed by mass spectrometry. We identified numerous phosphosites from HCN1 and HCN2 channels, representing a novel survey of phosphorylation sites within HCN channels. We found high levels of HCN channel phosphosite homology between humans and rats. We also identified a novel HCN1 channel phosphosite S791, which underwent significantly increased phosphorylation during the chronic epilepsy stage. Heterologous expression of a phosphomimetic mutant, S791D, replicated a hyperpolarizing shift in I(h) gating seen in neurons from chronically epileptic rats. These results show that HCN1 channel phosphorylation is altered in epilepsy and may be of pathogenic importance.

© 2016 by The Author(s). Asubset of potassium channels is regulated primarily by changes in the cytoplasmic concentration of ions, including calcium, sodium, chloride, and protons. The eight members of this subfamily were originally all designated as calcium-activated channels. More recent studies have clarified the gating mechanisms for these channels and have documented that not allmembers are sensitive to calcium. This article describes themolecular relationships between these channels and provides an introduction to their functional properties. It also introduces a new nomenclature that differentiates between calcium- and sodium-activated potassium channels.

Breathing must be tightly coordinated with other behaviours such as vocalization, swallowing, and coughing. These behaviours occur after inspiration, during a respiratory phase termed postinspiration. Failure to coordinate postinspiration with inspiration can result in aspiration pneumonia, the leading cause of death in Alzheimer's disease, Parkinson's disease, dementia, and other neurodegenerative diseases. Here we describe an excitatory network that generates the neuronal correlate of postinspiratory activity in mice. Glutamatergic-cholinergic neurons form the basis of this network, and GABA (γ-aminobutyric acid)-mediated inhibition establishes the timing and coordination relative to inspiration. We refer to this network as the postinspiratory complex (PiCo). The PiCo has autonomous rhythm-generating properties and is necessary and sufficient for postinspiratory activity in vivo.The PiCo also shows distinct responses to neuromodulators when compared to other excitatory brainstem networks. On the basis of the discovery of the PiCo, we propose that each of the three phases of breathing is generated by a distinct excitatory network: the pre-Bötzinger complex, which has been linked to inspiration; the PiCo, as described here for the neuronal control of postinspiration; and the lateral parafacial region (pF(L)), which has been associated with active expiration, a respiratory phase that is recruited during high metabolic demand.

In the mammalian cerebellum, deep cerebellar nuclear (DCN) cells convey all information from cortical Purkinje cells (PCs) to premotor nuclei and other brain regions. However, how DCN cells integrate inhibitory input from PCs with excitatory inputs from other sources has been difficult to assess, in part due to the large spatial separation between cortical PCs and their target cells in the nuclei. To circumvent this problem we have used a Cre-mediated genetic approach to generate mice in which channelrhodopsin-2 (ChR2), fused with a fluorescent reporter, is selectively expressed by GABAergic neurons, including PCs. In recordings from brain slice preparations from this model, mammalian PCs can be robustly depolarized and discharged by brief photostimulation. In recordings of postsynaptic DCN cells, photostimulation of PC axons induces a strong inhibition that resembles these cells’ responses to focal electrical stimulation, but without a requirement for the glutamate receptor blockers typically applied in such experiments. In this optogenetic model, laser pulses as brief as 1 ms can reliably induce an inhibition that shuts down the spontaneous spiking of a DCN cell for ∼50 ms. If bursts of such brief light pulses are delivered, a fixed pattern of bistable bursting emerges. If these pulses are delivered continuously to a spontaneously bistable cell, the immediate response to such photostimulation is inhibitory in the cell’s depolarized state and excitatory when the membrane has repolarized; a less regular burst pattern then persists after stimulation has been terminated. These results indicate that the spiking activity of DCN cells can be bidirectionally modulated by the optically activated synaptic inhibition of cortical PCs.

The slo3 gene encodes a K(+) channel found only in mammalian testis. This is in contrast to slo1, which is expressed in many tissues. Genes pertaining to male reproduction, especially those involved in sperm production, evolve morphologically and functionally much faster than their nonsexual counterparts. A comparison of SLO3 channel amino acid sequences from several species revealed a high degree of structural divergence relative to their SLO1 channel paralogues. To reveal any biophysical differences accompanying this rapid structural divergence, we analyzed the functional properties of SLO3 channels from two species, bovine and mouse. We observed several functional differences including voltage range of activation, kinetics, and pH sensitivity. Although SLO3 channel proteins from these two species lack conservation in many structural regions, we found that the first two of these three functional differences map to the same loop structure in their RCK1 (regulator of K(+) conductance 1) domain, which links the intermediate RCK1 subdomain to the C-terminal subdomain. We found that small structural changes in this region produce major changes in the voltage range of activation and kinetics. This rapidly evolving loop peptide shows the greatest length and sequence polymorphisms within RCK1 domains from many different species. In SLO3 channels this region may permit evolutionary changes that tune the gating properties in different species.

One of the largest components of the delayed outward current that is active under physiological conditions in many mammalian neurons, such as medium spiny neurons of the striatum and tufted-mitral cells of the olfactory bulb, has gone unnoticed and is the result of a Na(+)-activated K(+) current. Previous studies of K(+) currents in mammalian neurons may have overlooked this large outward component because the sodium channel blocker tetrodotoxin (TTX) is typically used in such studies. We found that TTX also eliminated this delayed outward component in rat neurons as a secondary consequence. Unexpectedly, we found that the activity of a persistent inward sodium current (persistent I(Na)) is highly effective at activating this large Na(+)-dependent (TTX sensitive) delayed outward current. Using siRNA techniques, we identified SLO2.2 channels as being carriers of this delayed outward current. These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the ∼90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict ∼11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during ∼350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

The chromanol 293B (293B, trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman) is a lead compound of potential class III antiarrhythmics that inhibit cardiac I(Ks) potassium channels. These channels are formed by the coassembly of KCNQ1 (Kv7.1, KvLQT1) and KCNE1 subunits. Although homomeric KCNQ1 channels are the principal molecular targets, entry of KCNE1 to the channel complex enhances the chromanol block. Because closely related neuronal KCNQ2 potassium channels are insensitive to the drug, we used KCNQ1/KCNQ2 chimeras to identify the binding site of the inhibitor. We localized the putative drug receptor to the H5 selectivity filter and the S6 transmembrane segment. Single residues affecting 293B inhibition were subsequently identified through systematic exchange of amino acids that were either different in KCNQ1 and KCNQ2 or predicted by a docking model of 293B in the open and closed conformation of KCNQ1. Mutant channel proteins T312S, I337V, and F340Y displayed dramatically lowered sensitivity to chromanol block. The predicted drug binding receptor lies in the inner pore vestibule containing the lower part of the selectivity filter, and the S6 transmembrane domain also reported to be important for binding of benzodiazepines. We propose that the block of the ion permeation pathway involves hydrophobic interactions with the S6 transmembrane residues Ile337 and Phe340, and stabilization of chromanol 293B binding through electrostatic interactions of its oxygen atoms with the most internal potassium ion within the selectivity filter.

Potassium-selective channels are the largest and most diverse group of ion channels, represented by some 70 known loci in the mammalian genome. The first cloned potassium channel gene was the Drosophila voltage-gated shaker channel, and this was rapidly followed by the identification of other

The human KCNQ gene family encodes potassium channels linked to several genetic syndromes including neonatal epilepsy, cardiac arrhythmia, and progressive deafness. KCNQ channels form M-type potassium channels, which are critical regulators of neuronal excitability that mediate autonomic responses, pain, and higher brain function. Fundamental mechanisms of the normal and abnormal cellular roles for these channels may be gained from their study in simple model organisms. Here we report that a multigene family of KCNQ-like channels is present in the nematode, Caenorhabditis elegans. We show that many aspects of the functional properties, tissue expression pattern, and modulation of these C. elegans channels are conserved, including suppression by the M1 muscarinic receptor. We also describe a conserved mechanism of modulation by diacylglycerol for a subset of C. elegans and vertebrate KCNQ/KQT channels, which is dependent upon the carboxyl-terminal domains of channel subunits and activated protein kinase C.

This summary article presents an overview of the molecular relationships among the voltage-gated sodium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the sodium channel family can be found athttp://www.iuphar-db.org/iuphar-ic/.

GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K + currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo . In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca 2+ and Cl - were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances.

The gating of different potassium channels depends on many diverse factors. We now report a unique example of a K+ channel with a Cl - dependence. The slo-2 gene was cloned from Caenorhabditis elegans and is widely expressed in both neurons and muscles; it was highly abundant, as suggested by its high representation in the C. elegans EST database. SLO-2, like its paralogue, SLO-1, was also dependent on Ca 2+. We show by site-directed mutagenesis that its requirements for both Cl- and Ca2+ are synergistic and associated with the same functional domain. SLO-2's dependence on Cl - implies that intracellular Cl- homeostasis may be important in regulating cellular excitability through this unusual K+ channel.

K+channels are key regulators of cellular excitability. Mutations that activate K+channels can lower cellular excitability, whereas those that inhibit K+channels may increase excitability. We show that theCaenorhabditis elegansegl-2gene encodes an eag K+channel and that a gain-of-function mutation inegl-2blocks excitation in neurons and muscles by causing the channel to open at inappropriately negative voltages. Tricyclic antidepressants reverseegl-2(gf)mutant phenotypes, suggesting that EGL-2 is a tricyclic target. We verified this by showing that EGL-2 currents are inhibited by imipramine. Similar inhibition is observed with the mouse homolog MEAG, suggesting that inhibition of EAG-like channels may mediate some clinical side effects of this class of antidepressants.

The wealth of accumulating data from the Caenorhabditis elegans genome sequencing project has rapidly accelerated the discovery of novel potassium channel genes and now places within reach the possibility of describing the total complement of potassium channels used by an individual species. Using annotated GenBank sequences, BLAST searches of unfinished sequences and degenerate oligonucleotide polymerase chain reaction (PCR) screens, we have identified and compiled genes for 38 C. elegans potassium channel and two cyclic nucleotide-gated cation channel subunits, representing eight conserved multigene families. Novel families of potassium channel genes were revealed, as well as conserved homologues of all known vertebrate families. Two separate families represent C. elegans homologues for human potassium channels recently implicated in hereditary long QT arrhythmias. Of particular note is an exceptionally large class of at least 23 genes with a novel subunit structure having two tandem 'P' domains; these channels may form as dimers in contrast to all other potassium channel types which form as tetramers. The 40 potassium channel genes are evenly distributed on all six C. elegans chromosomes, with the exception of the instances of gene clustering on the fifth and X chromosomes.

The molecular basis of general anesthetic action on membrane proteins that control ion transport is not yet understood. In a previous report (Covarrubias, M., and Rubin, E.(1993) Proc. Natl. Acad. Sci. 90, 6957-6960), we found that low concentrations of ethanol (17-170 mM) selectively inhibited a noninactivating cloned K channel encoded by Drosophila Shaw2. Here, we have conducted equilibrium dose-inhibition experiments, single channel recording, and mutagenesis in vitro to study the mechanism underlying the inhibition of Shaw2 K channels by a homologous series of n-alkanols (ethanol to 1-hexanol). The results showed that: (i) these alcohols inhibited Shaw2 whole-cell currents, the equilibrium dose-inhibition relations were hyperbolic, and competition experiments revealed the presence of a discrete site of action, possibly a hydrophobic pocket; (ii) this pocket may be part of the protein because n-alkanol sensitivity can be transferred to novel hybrid K channels composed of Shaw2 subunits and homologous ethanol-insensitive subunits; (iii) moreover, a hydrophobic point mutation within a cytoplasmic loop of an ethanol-insensitive K channel (human Kv3.4) was sufficient to allow significant inhibition by n-alkanols, with a dose-inhibition relation that closely resembled that of wild-type Shaw2 channels; and (iv) 1-butanol selectively inhibited long duration single channel openings in a manner consistent with a direct effect on channel gating. These results strongly suggest that a discrete site within the ion channel protein is the primary locus of alcohol and general anesthetic action.

The effect of protein kinase C (PKC) on rapid N-type inactivation of K+ channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K+ channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system.

High conductance, Ca(2+)-activated (BK-type) K+ channels from mouse (mSlo) and Drosophila (dSlo) differ in their functional properties but share a conserved core resembling voltage-gated K+ channels and a tail appended to the core by a nonconserved linker. We have found that the channel subunit is physically divisible into these two conserved domains and that the core determines such properties as channel open time, conductance, and, probably, voltage dependence, whereas the tail determines apparent Ca2+ sensitivity. Both domains are required for function. We demonstrated the different roles of the core and tail by taking advantage of the functional differences between mSlo and dSlo. Heterologous pairing of cores and tails from mSlo and dSlo showed that single-channel properties were always characteristic of the core species, but that apparent Ca2+ sensitivity was adjusted up or down depending on the species of the tail. Thus, the tail is implicated in the Ca(2+)-sensing role of BK channels.

The molecular genetic approach to studying K + channels has revealed that at least four subfamilies of voltage-gated K + channels originally discovered in Drosophila are conserved in mice and humans. This conservation of the K + channel subfamilies Shaker, Shal, Shab, and Shaw suggests that not only the broad outlines of membrane electrical properties but also many molecular details as well evolved in the parent species ancestral to both invertebrate and vertebrate life. Shaker, Shal, Shab, and Shaw K + channels have similar structures, but appear to be independent channel systems: when co-expressed in Xenopus oocytes, all four function independently. These four K + channel subfamilies may be part of an essential ‘set' of excitable channels required by most nervous systems. The task now remaining is to understand the functions of each member of the set.

Although many K+ channel genes encoding homologous subunits have been cloned, a central question remains: how do these subunits associate to produce the diversity of K+ currents observed in living cells? Previous work has shown that different subunits encoded by the Shaker gene subfamily are able to form heteromultimers, which add to the diversity of currents. However, the unrestrained mixing of subunits from all genes to form hybrid channels would be undesirable for some cells that clearly require functionally discrete K+ currents. We show that Drosophila Shaker, Shal, Shab, and Shaw subunits form functional homomultimers, but that a molecular barrier to heteropolymerization is present. Coexpression of all four K+ channel systems does not alter their individual properties in any way. These experiments also demonstrate that multiple, independent A-current systems together with multiple, independent delayed rectifier systems can coexist in single cells.

High-conductance, 'big' potassium (BK) channels encoded by the Slo gene family are among the largest and most complex of the extended family of potassium channels. The family of SLO channels apparently evolved from voltage-dependent potassium channels, but acquired a large conserved carboxyl extension, which allows channel gating to be altered in response to the direct sensing of several different intracellular ions, and by other second-messenger systems, such as those activated following neurotransmitter binding to G-protein-coupled receptors (GPCRs). This versatility has been exploited to serve many cellular roles, both within and outside the nervous system.

Slick (Slo2.1) and Slack (Slo2.2) are two novel members of the mammalianSlopotassium channel gene family that may contribute to the resting potentials of cells and control their basal level of excitability. Slo2 channels have sensors that couple channel activity to the intracellular concentrations of Na+and Cl−ions (Yuan et al., 2003). We now report that activity of both Slo2 channels is controlled by neuromodulators through Gαq-protein coupled receptors (GqPCRs) (the M1muscarinic receptor and the mGluR1 metabotropic glutamate receptor). Experiments coexpressing channels and receptors inXenopusoocytes show that Slo2.1 and Slo2.2 channels are modulated in opposite ways: Slo2.1 is strongly inhibited, whereas Slo2.2 currents are strongly activated through GqPCR stimulation. Differential regulation involves protein kinase C (PKC); application of the PKC activator PMA, to cells expressing channels but not receptors, inhibits Slo2.1 whole-cell currents and increases Slo2.2 currents. Synthesis of a chimera showed that the distal carboxyl region of Slo2.1 controls the sensitivity of Slo2.1 to PMA. Slo2 channels have widespread expression in brain (Bhattacharjee et al., 2002, 2005). Using immunocytochemical techniques, we show coexpression of Slo2 channels with the GqPCRs in cortical and hippocampal brain sections and in cultured hippocampal neurons. The differential control of these novel channels by neurotransmitters may elicit long-lasting increases or decreases in neuronal excitability and, because of their widespread distribution, may provide a mechanism to activate or repress electrical activity in many systems of the brain.

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Na + -activated potassium channels (K Na ) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. We now report that K Na is encoded by the rSlo2 gene (also called Slack ), the mammalian ortholog of slo-2 in C. elegans . rSlo2 , heterologously expressed, shares many properties of native K Na including activation by intracellular Na + , high conductance, and prominent subconductance states. In addition to activation by Na + , we report that rSLO-2 channels are cooperatively activated by intracellular Cl − , similar to C. elegans SLO-2 channels. Since intracellular Na + and Cl − both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis revealed that the SLO-2 current is a major component of the delayed rectifier. We demonstrate in C. elegans that slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily.

The relationship between the genome and the evolution of the nervous system may differ between an animal like C. elegans with 302 neurons, and mammals with tens of billions of neurons. Here we report that a class of nonconserved potassium channels highly expanded in C. elegans may play a special role in the evolution of its nervous system. The C. elegans genome contains an extended gene family of potassium channels whose members fall into two evolutionary divergent classes. One class constitutes an ancient conserved “set” of K + channels with orthologues in both humans and Drosophila 1. , 24. and a second larger class made up of rapidly evolving genes unique to C. elegans . 24 Chief among this second class are novel potassium channels having four transmembrane domains per subunit 6. , 8. , 14. , 20. that function as regulated leak conductances to modulate cell electrical excitability. This inventory of novel potassium channels is far larger in C. elegans than in humans or Drosophila . We found that, unlike conserved channel genes, the majority of these genes are expressed in very few cells. We also identified DNA enhancer elements associated with these genes that direct gene expression to individual neurons. We conclude that C. elegans may maintain an exceptionally large inventory of these channels (as well as ligand-gated channels 1 ) as an adaptive mechanism to “fine tune” individual neurons, making the most of its limited circuitry.

As many as 50 genes in the C. elegans genome may encode K+ channels belonging to the novel structural class of two-pore (4TM) channels. Many 4TM channels can be grouped into channel subfamilies. We analyzed 4TM channels in C. elegans using methods made possible by having complete genomic sequence. Two genes were chosen for comprehensive analysis, n2P16 and n2P17. By comparing the pattern of conservation in genomic DNA sequences between C. elegans and a closely related species, C. briggsae, we were able to identify all coding regions and predict the gene structure for these two genes. Given the extent of the 4TM channel family, we were surprised to discover that n2P17 produced at least six alternative transcripts encoding a constant central region and variable amino- and carboxyl-termini. Blocks of highly conserved DNA sequences in noncoding regions were also apparent and most likely confer important regulatory functions. The interspecies comparison of the deduced channel proteins revealed that the extracellular loop between M1 and P1 is an apparent hot spot for evolutionary change in both channels. This contrasts with the membrane-spanning domains that are highly conserved. Analysis of intron positions for 36 channels revealed that introns are frequently present at an identical position within the pore region, but very few are located in membrane-spanning domains.

Publisher Summary This chapter discusses the impact of the Caenorhabditis elegans genome project on potassium channel biology. The analysis of potassium channels in C. elegans from a “functional genomics” perspective has many unique aspects: C. elegans has a genome size of approximately one hundred million base pairs. The availability of the entire DNA sequence of the C. elegans genome provides the first glimpse of the entire set of K + channels in one animal. This nearly complete picture of potassium channel diversity is revealed in an unbiased way because no particular cell, tissue type, or abundance class is favored. In addition to the primary sequence of a gene product and all alternative forms, a complete genomic sequence allows addressing a host of questions that were previously inaccessible. These questions include the genetic determinants of tissue specificity such as enhancers, promoters, and sequences that determine splice-site selection, and other transcribed but nontranslated regions that may control message transport, localization, or lifetime.

Potassium channels have evolved to play specialized roles in both excitable and inexcitable tissues. Here we describe the cloning and expression of Slo3, a novel potassium channel abundantly expressed in mammalian spermatocytes. Slo3 represents a new and unique type of potassium channel regulated by both intracellular pH and membrane voltage. Reverse transcription-polymerase chain reaction, Northern analysis, and in situ hybridization show that Slo3 is primarily expressed in testis in both mouse and human. Because of its sensitivity to both pH and voltage, Slo3 could be involved in sperm capacitation and/or the acrosome reaction, essential steps in fertilization where changes in both intracellular pH and membrane potential are known to occur. The protein sequence of mSlo3 (the mouse Slo3 homologue) is similar to Slo1, the large conductance, calcium- and voltage-gated potassium channel. These results suggest that Slo channels comprise a multigene family, defined by a combination of sensitivity to voltage and a variety of intracellular factors. Northern analysis from human testis indicates that a Slo3 homologue is present in humans and conserved with regard to sequence, transcript size, and tissue distribution. Because of its high testis-specific expression, pharmacological agents that target human Slo3 channels may be useful in both the study of fertilization as well as in the control or enhancement of fertility.

Complementary DNAs (cDNAs) from mSlo, a gene encoding calcium-activated potassium channels, were isolated from mouse brain and skeletal muscle, sequenced, and expressed in Xenopus oocytes. The mSlo-encoded channel resembled "maxi" or BK (high conductance) channel types; single channel conductance was 272 picosiemens with symmetrical potassium concentrations. Whole cell and single channel currents were blocked by charybdotoxin, iberiotoxin, and tetraethylammonium ion. A large number of variant mSlo cDNAs were isolated, indicating that several diverse mammalian BK channel types are produced by a single gene.

The authors would like to apologize for failing to correct an error in their description of the two-step PCR used in their protocol, which reverses the order of the primer pairs used for the first and second rounds of PCR. This error is found in two locations in the manuscript: On page 2, ‘Two rounds of amplification were used, an initial round of 35 cycles with the inner primer set, followed by a second round of 35 cycles with the corresponding outer primer set.’ should be changed to ‘Two rounds of amplification were used, an initial round of 35 cycles with the outer primer set, followed by a second round of 35 cycles with the corresponding inner primer set.’ On page 3, in the legend for Figure 1, ‘Second round PCRs (outer primers) were performed with a dilution of the first round (inner primers) reaction.’ should be changed to ‘Second round PCRs (inner primers) were performed with a dilution of the first round (outer primers) reaction.’ The correct order of primer sets that they used for two-step PCR is outer primer set for the first round, followed by inner primer set for the second round. This descriptive error does not alter any of the conclusions of the paper. The authors regret any inconvenience or misunderstanding that their error may have caused.

Mutant flies in which the gene coding for the Shaker potassium channel is deleted still have potassium currents similar to those coded by the Shaker gene. This suggests the presence of a family of Shaker-like genes in Drosophila. By using a Shaker complementary DNA probe and low-stringency hybridization, three additional family members have now been isolated, Shab, Shaw, and Shal. The Shaker family genes are not clustered in the genome. The deduced proteins of Shab, Shaw, and Shal have high homology to the Shaker protein; the sequence identity of the integral membrane portions is greater than 50 percent. These genes are organized similarly to Shaker in that only a single homology domain containing six presumed membrane-spanning segments common to all voltage-gated ion channels is coded by each messenger RNA. Thus, potassium channel diversity could result from an extended gene family, as well as from alternate splicing of the Shaker primary transcript.

Mutations in the C. elegans egl-36 gene result in defective excitation of egg-laying and enteric muscles. Dominant gain-of-function alleles inhibit enteric and egg-laying muscle contraction, whereas a putative null mutation has no observed phenotype. egl-36 encodes a Shaw-type (Kv3) voltage-dependent potassium channel subunit. In Xenopus oocytes, wild-type egl-36 expresses noninactivating channels with slow activation kinetics. One gain-of-function mutation causes a single amino acid substitution in S6, and the other causes a substitution in the cytoplasmic amino terminal domain. Both mutant alleles produce channels dramatically shifted in their midpoints of activation toward hyperpolarized voltages. An egl-36::gfp fusion is expressed in egg-laying muscles and in a pair of enteric muscle motor neurons. The mutant egl-36 phenotypes can thus be explained by expression in these cells of potassium channels that are inappropriately opened at hyperpolarized potentials, causing decreased excitability due to increased potassium conductance.

The integral membrane protein composing the voltage-sensitive sodium channel is a bagel-like structure (Fig. 1) with a central pore that is highly selective for sodium ions. When the electric field across a cell's membrane becomes depolarized, the pore of the channel opens to allow sodium ions to flow down their electrochemical gradient into the cell 1 . Voltage-gating, a common feature of all voltage-activated channels, is an essential functional property that is only now being understood in terms of the structural features of the protein.

In the membrane of the flight muscle cells of developing Drosophila a large calcium-sensitive potassium current, IKc, was found. It was present before the development of voltage-activated potassium channels and seems to be the first potassium current to develop in the membrane. Also present in these early cells were large numbers of occult (hidden) calcium channels, which remained inactive until the end of pupal development. These inactive calcium channels could be made to function by injecting adenosine triphosphate or ethyleneglycol tetraacetic acid into the early cells. IKc has kinetic properties resembling the later developing voltage-sensitive current IKv, and is distinct from the fast, transient calcium-dependent outward current IAc, which appears much later in development. IAc closely resembles the voltage-sensitive current IAv, also present in these cells. Thus, both of the voltage-sensitive potassium channel types, IAv and IKv, have similar calcium-sensitive counterparts, IAc and IKc, that are present in the same cells.

The deduced amino acid sequence of a Drosophila gene isolated with a vertebrate sodium channel complementary DNA probe revealed an organization virtually identical to the vertebrate sodium channel protein; four homologous domains containing all putative membrane-spanning regions are repeated in tandem with connecting linkers of various sizes. All areas of the protein presumed to be critical for channel function show high evolutionary conservation. These include those proposed to function in voltage-sensitive gating, inactivation, and ion selectivity. All 24 putative gating charges of the vertebrate protein are in identical positions in the Drosophila gene. Ten introns interrupt the coding regions of the four homology units; introns with positions conserved among homology units bracket a region hypothesized to be the selectivity filter for the channel. The Drosophila gene maps to the right arm of the second chromosome in region 60D-E. This position does not coincide with any known mutations that confer behavioral phenotypes, but is close to the seizure locus (60A-B), which has been hypothesized to code for a voltage-sensitive sodium channel.

Ion channels are the "transistors" (electronic switches) of the brain that generate and propagate electrical signals in the aqueous environment of the brain and nervous system. Potassium channels are particularly important because, not only do they shape dynamic electrical signaling, they also set the resting potentials of almost all animal cells. Without them, animal life as we know it would not exist, much less higher brain function. Until the completion of the C. elegans genome sequencing project the size and diversity of the potassium channel extended gene family was not fully appreciated. Sequence data eventually revealed a total of approximately 70 genes encoding potassium channels out of the more than 19,000 genes in the genome. This seemed to be an unexpectedly high number of genes encoding potassium channels for an animal with a small nervous system of only 302 neurons. However, it became clear that potassium channels are expressed in all cell types, not only neurons, and that many cells express a complex palette of multiple potassium channels. All types of potassium channels found in C. elegans are conserved in mammals. Clearly, C. elegans is "simple" only in having a limited number of cells dedicated to each organ system; it is certainly not simple with respect to its biochemistry and cell physiology.

Genes pertaining to male reproduction, especially those involved in sperm production, morphologically and functionally evolve much faster than their non-sexual counterparts. SLO3 is an especially intriguing example of such a rapidly evolving gene. The SLO3 gene encodes a K+ channel which is expressed only in mammalian sperm and is evolving much faster than its close paralogue SLO1 which is expressed in brain and other organs. We cloned the bovine orthologue of SLO3 (bSLO3) and compared its primary sequence and functional properties to its mouse orthologue (mSLO3) which we previously cloned. A comparison of bSLO3 and mSLO3 primary sequences showed far less conservation than for SLO1 proteins in mouse and bovine species. Functionally, bSLO3 and mSLO3 also differ markedly with respect to their voltage range of activation, their ion selectivity, and their activation kinetics. Remarkably, although there are many regions of low conservation between bSLO3 and mSLO3 proteins, we found that all of the different functional properties that we measured map to a small region of low conservation in the RCK1 domain. One or more of these different functional properties may reflect differences in the resting membrane potentials of sperm in bovine and mouse species. This work was supported by National Institute of Health grants 1R21HD056444-01A1 to C.M.S. and R24 RR017342-01 and R01 GM067154-01A1 to L.S.

The Hodgkin-Huxley model of delayed repolarization of the sodium-dependent action potential assigns a voltage-dependent delayed rectifier K+ current for repolarization. However, here we show that one of the largest components of the delayed rectifier current in many mammalian neurons has gone unnoticed and is due to a Na+-activated-K+-current/Persistent-Na+-current coupled system. Previous studies of potassium conductances in mammalian neurons may have overlooked this large outward component because the sodium channel blocker TTX is typically used in such studies; we find that, in addition to blocking sodium currents, TTX also eliminates this delayed rectifier component as a secondary consequence. We unexpectedly found that the activity of the persistent inward sodium current (persistent INa) at cell resting potentials is the essential factor in activating the Na+-dependent (TTX-sensitive) delayed rectifier current. The persistent INa apparently maintains Na+-activated K+ channels in a “primed” state so that, upon depolarization, they carry a delayed outward conductance. Persistent INa appears to raise the local concentration of Na+ in the vicinity of Na+-activated K+ channels to higher levels than that of the bulk cytosol, possibly because of a submembrane diffusion-restricted space variously referred to in the literature as an “unstirred” layer” or “fuzzy” space”. We showed that “depleting” or “filling” this diffusion-restricted space by the action of persistent INa requires several seconds at cell resting potentials. Using siRNA techniques we identified SLO2.2 (Slack) channels as carriers of the Na+-dependent delayed rectifier current. These findings of a previously unseen K+ conductance involving a finely tuned partnership linking persistent INa and Na+-activated K+ channels, have far reaching implications for many neurons of the mammalian brain. Studies of “up-down” states of neuronal excitability, spike adaptation, synaptic integration, and other aspects of neuronal physiology may have to be reexamined taking this system into account.