LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology.

Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryoelectron microscopy (cryo-EM) structures of nonnative LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c-/- HCT116 cells are poorly activated by low intracellular ionic strength (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A, allowing characterization of pore properties. VRACs are strongly inhibited by 10 µM 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, an LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ∼90% by 10 µM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ dependent. At normal intracellular µ, LRRC8A + LRRC8C heteromers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heteromeric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homomeric channels.

CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor.

Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 (LRRC8) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate Gq-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- Gq-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.

The Skp1 Homologs SKR-1/2 Are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK.

SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.

Regulatory Conformational Coupling between CLC Anion Channel Membrane and Cytoplasmic Domains.

CLC anion channels are homodimeric proteins. Each subunit is comprised of 18 α-helices designated "A-R" and an intracellular carboxy-terminus containing two cystathionine-ß-synthase (CBS1 and CBS2) domains. Conformational coupling between membrane and intracellular domains via poorly understood mechanisms is required for CLC regulation. The activity of the C. elegans CLC channel CLH-3b is reduced by phosphorylation of a carboxy-terminus "activation domain," which disrupts its interaction with CBS domains. CBS2 interfaces with a short intracellular loop, the H-I loop, connecting membrane helices H and I. Alanine mutation of a conserved H-I loop tyrosine residue, Y232, prevents regulation demonstrating that the loop functions to couple phosphorylation-dependent CBS domain conformational changes to channel membrane domains. To gain further insight into the mechanisms of this coupling, we mutated conserved amino acid residues in membrane helices H and I. Only mutation of the H-helix valine residue V228 to leucine prevented phosphorylation-dependent channel regulation. Structural and functional studies of other CLC proteins suggest that V228 may interact with Y529, a conserved R-helix tyrosine residue that forms part of the CLC ion conduction pathway. Mutation of Y529 to alanine also prevented CLH-3b regulation. Intracellular application of the sulfhydryl reactive reagent MTSET using CLH-3b channels engineered with single-cysteine residues in CBS2 indicate that V228L, Y529A, and Y232A disrupt putative regulatory intracellular conformational changes. Extracellular Zn2+ inhibits CLH-3b and alters the effects of intracellular MTSET on channel activity. The effects of Zn2+ are disrupted by V228L, Y529A, and Y232A. Collectively, our findings indicate that there is conformational coupling between CBS domains and the H and R membrane helices mediated by the H-I loop. We propose a simple model by which conformational changes in H and R helices mediate CLH-3b regulation by activation domain phosphorylation.

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-ß-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways.

The voltage-gated anion channels encoded by clh-3 regulate egg laying in C. elegans by modulating motor neuron excitability.

CLC-2 is a hyperpolarization-activated, inwardly rectifying chloride channel. Although the properties of the CLC-2 channel have been well characterized, its function in vivo is not well understood. We have found that channels encoded by the Caenorhabditis elegans CLC-2 homolog clh-3 regulate the activity of the spontaneously active hermaphrodite-specific neurons (HSNs), which control the egg-laying behavior. We identified a gain-of-function mutation in clh-3 that increases channel activity. This mutation inhibits egg laying and inhibits HSN activity by decreasing its excitability. Conversely, loss-of-function mutations in clh-3 lead to misregulated egg laying and an increase in HSN excitability, indicating that these channels modulate egg laying by limiting HSN excitability. clh-3-encoded channels are not required for GABAA-receptor-mediated inhibition of the HSN. However, they require low intracellular chloride for HSN inhibition, indicating that they inhibit excitability directly by mediating chloride influx. This mechanism of CLH-3-dependent modulation may be conserved in other neurons in which the driving force favors chloride influx.

Regulatory phosphorylation induces extracellular conformational changes in a CLC anion channel.

CLH-3b is a CLC-1/2/Ka/Kb channel homolog activated by meiotic cell cycle progression and cell swelling. Channel inhibition occurs by GCK-3 kinase-mediated phosphorylation of serine residues on the cytoplasmic C-terminus linker connecting CBS1 and CBS2. Two conserved aromatic amino acid residues located on the intracellular loop connecting membrane helices H and I and α1 of CBS2 are required for transducing phosphorylation changes into changes in channel activity. Helices H and I form part of the interface between the two subunits that comprise functional CLC channels. Using a cysteine-less CLH-3b mutant, we demonstrate that the sulfhydryl reagent reactivity of substituted cysteines at the subunit interface changes dramatically during GCK-3-mediated channel inhibition and that these changes are prevented by mutation of the H-I loop/CBS2 α1 signal transduction domain. We also show that GCK-3 modifies Zn(2+) inhibition, which is thought to be mediated by the common gating process. These and other results suggest that phosphorylation of the cytoplasmic C-terminus inhibits CLH-3b by inducing subunit interface conformation changes that activate the common gate. Our findings have important implications for understanding CLC regulation by diverse signaling mechanisms and for understanding the structure/function relationships that mediate intraprotein communication in this important family of Cl(-) transport proteins.

Changes in translation rate modulate stress-induced damage of diverse proteins.

Proteostasis is the maintenance of the proper function of cellular proteins. Hypertonic stress disrupts proteostasis and causes rapid and widespread protein aggregation and misfolding in the nematode Caenorhabditis elegans. Optimal survival in hypertonic environments requires degradation of damaged proteins. Inhibition of protein synthesis occurs in response to diverse environmental stressors and may function in part to minimize stress-induced protein damage. We recently tested this idea directly and demonstrated that translation inhibition by acute exposure to cycloheximide suppresses hypertonicity-induced aggregation of polyglutamine::YFP (Q35::YFP) in body wall muscle cells. In this article, we further characterized the relationship between protein synthesis and hypertonic stress-induced protein damage. We demonstrate that inhibition of translation reduces hypertonic stress-induced formation and growth of Q35::YFP, Q44::YFP, and α-synuclein aggregates; misfolding of paramyosin and ras GTPase; and aggregation of multiple endogenous proteins expressed in diverse cell types. Activation of general control nonderepressible-2 (GCN-2) kinase signaling during hypertonic stress inhibits protein synthesis via phosphorylation of eukaryotic initiation factor-2α (eIF-2α). Inhibition of GCN-2 activation prevents the reduction in translation rate and greatly exacerbates the formation and growth of Q35::YFP aggregates and the aggregation of endogenous proteins. The current studies together with our previous work provide the first direct demonstration that hypertonic stress-induced reduction in protein synthesis minimizes protein aggregation and misfolding. Reduction in translation rate also serves as a signal that activates osmoprotective gene expression. The cellular proteostasis network thus plays a critical role in minimizing hypertonic stress-induced protein damage, in degrading stress-damaged proteins, and in cellular osmosensing and signaling.

Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly.

Volume-regulated anion channels (VRACs) mediate volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL125) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL125) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.

CLC anion channel regulatory phosphorylation and conserved signal transduction domains.

The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a ∼176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first α-helix (α1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 α1.

Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation.

Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-ß-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity.

GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling.

Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes.

Cell physiology at the Mount Desert Island Biological Laboratory: a brief look back and forward.

The Mount Desert Island Biological Laboratory (MDIBL) has played important roles in the development of modern physiological concepts and tools, particularly in the fields of kidney and epithelial cell physiology. Over the last decade, MDIBL has undergone remarkable growth and evolution. This article will briefly review MDIBL's past and outline its future directions. It is hoped that this overview will renew and stimulate interest in MDIBL and, in particular, will encourage an even wider community of physiologists to participate in its ongoing growth and development.

C. elegans STK39/SPAK ortholog-mediated inhibition of ClC anion channel activity is regulated by WNK-independent ERK kinase signaling.

Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl(-) cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes.

Hypertonic stress induces rapid and widespread protein damage in C. elegans.

Proteostasis is defined as the homeostatic mechanisms that maintain the function of all cytoplasmic proteins. We recently demonstrated that the capacity of the proteostasis network is a critical factor that defines the limits of cellular and organismal survival in hypertonic environments. The current studies were performed to determine the extent of protein damage induced by cellular water loss. Using worm strains expressing fluorescently tagged foreign and endogenous proteins and proteins with temperature-sensitive point mutations, we demonstrate that hypertonic stress causes aggregation and misfolding of diverse proteins in multiple cell types. Protein damage is rapid. Aggregation of a polyglutamine yellow fluorescent protein reporter is observable with <1 h of hypertonic stress, and aggregate volume doubles approximately every 10 min. Aggregate formation is irreversible and occurs after as little as 10 min of exposure to hypertonic conditions. To determine whether endogenous proteins are aggregated by hypertonic stress, we quantified the relative amount of total cellular protein present in detergent-insoluble extracts. Exposure for 4 h to 400 mM or 500 mM NaCl induced a 55-120% increase in endogenous protein aggregation. Inhibition of insulin signaling or acclimation to mild hypertonic stress increased survival under extreme hypertonic conditions and prevented aggregation of endogenous proteins. Our results demonstrate that hypertonic stress causes widespread and dramatic protein damage and that cells have a significant capacity to remodel the network of proteins that function to maintain proteostasis. These findings have important implications for understanding how cells cope with hypertonic stress and other protein-damaging stressors.

Phosphatidylinositol 4,5-bisphosphate and loss of PLCgamma activity inhibit TRPM channels required for oscillatory Ca2+ signaling.

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) oscillations that control muscle contractions required for defecation. Two highly Ca(2+)-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLCgamma in a common signaling pathway that regulates IP(3)-dependent intracellular Ca(2+) release. A second PLC, PLCbeta, is also required for IP(3)-dependent Ca(2+) oscillations, but functions in an independent signaling mechanism. PLCgamma generates IP(3) that regulates IP(3) receptor activity. We demonstrate here that PLCgamma via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) also regulates GON-2/GTL-1 function. Knockdown of PLCgamma but not PLCbeta activity by RNA interference (RNAi) inhibits channel activity approximately 80%. Inhibition is fully reversed by agents that deplete PIP(2) levels. PIP(2) added to the patch pipette has no effect on channel activity in PLCgamma RNAi cells. However, in control cells, 10 microM PIP(2) inhibits whole cell current approximately 80%. Channel inhibition by phospholipids is selective for PIP(2) with an IC(50) value of 2.6 microM. Elevated PIP(2) levels have no effect on channel voltage and Ca(2+) sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP(2) by PLCgamma functions in the activation of both the IP(3) receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca(2+) influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP(3) receptor function. PIP(2)-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca(2+) influx with PLCgamma and IP(3) receptor activity as well as intracellular Ca(2+) store depletion.

Cell cycle- and swelling-induced activation of a Caenorhabditis elegans ClC channel is mediated by CeGLC-7alpha/beta phosphatases.

ClC voltage-gated anion channels have been identified in bacteria, yeast, plants, and animals. The biophysical and structural properties of ClCs have been studied extensively, but relatively little is known about their precise physiological functions. Furthermore, virtually nothing is known about the signaling pathways and molecular mechanisms that regulate channel activity. The nematode Caenorhabditis elegans provides significant experimental advantages for characterizing ion channel function and regulation. We have shown previously that the ClC Cl- channel homologue CLH-3 is expressed in C. elegans oocytes, and that it is activated during meiotic maturation and by cell swelling. We demonstrate here that depletion of intracellular ATP or removal of Mg2+, experimental maneuvers that inhibit kinase function, constitutively activate CLH-3. Maturation- and swelling-induced channel activation are inhibited by type 1 serine/threonine phosphatase inhibitors. RNA interference studies demonstrated that the type 1 protein phosphatases CeGLC-7alpha and beta, both of which play essential regulatory roles in mitotic and meiotic cell cycle events, mediate CLH-3 activation. We have suggested previously that CLH-3 and mammalian ClC-2 are orthologues that play important roles in heterologous cell-cell interactions, intercellular communication, and regulation of cell cycle-dependent physiological processes. Consistent with this hypothesis, we show that heterologously expressed rat ClC-2 is also activated by serine/threonine dephosphorylation, suggesting that the two channels have common regulatory mechanisms.

A 30-year journey from volume-regulated anion currents to molecular structure of the LRRC8 channel.

The swelling-activated anion channel VRAC has fascinated and frustrated physiologists since it was first described in 1988. Multiple laboratories have defined VRAC's biophysical properties and have shown that it plays a central role in cell volume regulation and possibly other fundamental physiological processes. However, confusion and intense controversy surrounding the channel's molecular identity greatly hindered progress in the field for >15 yr. A major breakthrough came in 2014 with the demonstration that VRAC is a heteromeric channel encoded by five members of the Lrrc8 gene family, Lrrc8A-E. A mere 4 yr later, four laboratories described cryo-EM structures of LRRC8A homomeric channels. As the melee of structure/function and physiology studies begins, it is critical that this work be framed by a clear understanding of VRAC biophysics, regulation, and cellular physiology as well as by the field's past confusion and controversies. That understanding is essential for the design and interpretation of structure/function studies, studies of VRAC physiology, and studies aimed at addressing the vexing problem of how the channel detects cell volume changes. In this review we discuss key aspects of VRAC biophysics, regulation, and function and integrate these into our emerging understanding of LRRC8 protein structure/function.

A primary culture system for functional analysis of C. elegans neurons and muscle cells.

C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.