Mechanisms Of Channel Regulation Research Articles

Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

Potassium channels in the heart: structure, function and regulation.

This paper is the outcome of the fourth UC Davis Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias Symposium, a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2016 symposium was 'K+ Channels and Regulation'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies and challenges on the topic of cardiac K+ channels. This paper summarizes the topics of formal presentations and informal discussions from the symposium on the structural basis of voltage-gated K+ channel function, as well as the mechanisms involved in regulation of K+ channel gating, expression and membrane localization. Given the critical role for K+ channels in determining the rate of cardiac repolarization, it is hardly surprising that essentially every aspect of K+ channel function is exquisitely regulated in cardiac myocytes. This regulation is complex and highly interrelated to other aspects of myocardial function. K+ channel regulatory mechanisms alter, and are altered by, physiological challenges, pathophysiological conditions, and pharmacological agents. An accompanying paper focuses on the integrative role of K+ channels in cardiac electrophysiology, i.e. how K+ currents shape the cardiac action potential, and how their dysfunction can lead to arrhythmias, and discusses K+ channel-based therapeutics. A fundamental understanding of K+ channel regulatory mechanisms and disease processes is fundamental to reveal new targets for human therapy.

Novel BK channel regulatory mechanism by protein kinase C in guinea pig urinary bladder smooth muscle (865.1)

Large conductance Ca2+‐activated K+ (BK) channels are critical regulators of urinary bladder smooth muscle (UBSM) function. Here, we provide a novel mechanistic insight into BK channel regulation by protein kinase C (PKC) in UBSM. Voltage‐clamp experiments showed that pharmacological activation of PKC with phorbol 12‐myristate 13‐acetate (PMA), inhibited the spontaneous transient BK currents (TBKCs) in native freshly‐isolated guinea pig UBSM cells. Current‐clamp recordings revealed that PMA significantly depolarized UBSM membrane potential, and reduced the spontaneous transient hyperpolarizations in UBSM cells. The PMA inhibitory effects on UBSM membrane potential were abolished by the selective BK channel inhibitor paxilline and were not observed with its inactive analog, 4‐alpha‐PMA. PMA did not affect the amplitude of the whole cell steady‐state BK current or single BK channel open probability (recorded in cell‐attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PMA elevated the intracellular Ca2+ levels in UBSM cells, and increased spontaneous phasic and nerve‐evoked contractions of UBSM isolated strips. The results support the concept that PKC activation leads to a reduction in BK channel activity in UBSM via a Ca2+‐dependent mechanism, thus increasing UBSM contractility.Grant Funding Source: Supported by R01 DK084284 to G. V. Petkov

Use-Dependent Activation of Kv1.2 Channel Complexes

In excitable cells, ion channels are often challenged by rapid and repetitive stimuli. Ion channel responses to repetitive stimulation shape cellular behaviors by regulating the duration and termination of bursting action potentials. Therefore, we have investigated the regulation of voltage-gated potassium (Kv) channels under high frequency stimuli, with a particular focus on the canonical delayed rectifier Kv1.2. Previous reports have demonstrated a unique prepulse potentiation behavior that is observed in Kv1.2 channels expressed in mammalian cell lines. In this study, we demonstrate that this prepulse potentiation enables Kv1.2 channels to exhibit marked use-dependent activation, with trains of brief depolarizations causing dramatic increases in elicited current. This property arises from a stabilization of the channel open state in potentiated channels by ∼2.5 kcal/mol, reflecting a 28 ± 1 mV leftward shift in activation V1/2 after channel potentiation, and a marked acceleration of channel activation. Importantly, Kv subunits can assemble into heteromeric channels, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that although other Kv1 channel types are not prone to the use-dependent activation observed for Kv1.2, this property is conferred to other Kv1 subunits when they co-assemble with Kv1.2 in heteromeric channels. Our observations suggest a unique role for Kv1.2 subunits as potential suppressive components of heteromeric Kv1 channels, and describe a novel mechanism of channel regulation that will influence channel activity during bursts of cellular electrical activity. These findings illustrate that the functional output of heteromeric Kv channels integrates the biophysical properties and signaling sensitivities of their component subunits. We highlight the importance of expanding biophysical studies of Kv channels to better understand interactions between different subunit types in heteromeric complexes.

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Modification of glycan chains as a mechanism to regulate SOCE (P1156)

Abstract T cell receptor mediated activation requires the influx of calcium from the extracellular medium. Here, the endoplasmic reticulum (ER) Ca2+ sensor STIM1 reports the decrease in luminal [Ca2+] caused by IP3 receptor activity, clusters and directly binds and activates Orai1 channels, providing Ca2+ influx (store-operated, SOCE) essential for immune cell activation. We are interested in the regulation and modulation of this pathway. Because alterations of glycosylation of several T cell epitopes are implicated in immunsenescence and cancer, we investigated N-glycosylation site mutants of STIM1 and Orai1 and describe a STIM1 mutant (NN/DQ) which results in a strong gain-of-function. The mutant leads to a significant increase in the number of active channels despite a concomitant decrease in Orai1 protein, uncovering a novel CRAC channel regulatory mechanism. While we have already shown that the STIM1 mutant phenotype is mainly due to an increased rate of STIM1 oligomerization, we now investigate the specificity of this effect by analysing Ca2+ influx mediated by Orai2 and Orai3 and also started delineating the mechanism of Orai1 protein reduction. In addition, we are investigating the complex glycosylation pattern of primary human T cells and its effect on SOCE. Our findings indicate that the cell type specific repertoire of N-glycoslytransferases modifies the influence of the Orai1 glycan chains on Ca2+ influx.

Mechanical regulation of native and the recombinant calcium channel

L-type calcium channels are modulated by a host of mechanisms that include voltage, calcium ions (Ca2+ dependent inactivation and facilitation), cytosolic proteins (CAM, CAMKII, PKA, PKC, etc.), and oxygen radicals. Here we describe yet another Ca2+ channel regulatory mechanism that is induced by pressure-flow (PF) forces of ∼25dyn/cm2 producing 35–60% inhibition of channel current. Only brief periods (300ms) of such PF pulses were required to suppress reversibly the current. Recombinant Ca2+ channels (α1c77/β2a/α2δ and α1c77/β1/α2δ), expressed in HEK293 cells, were similarly suppressed by PF pulses. To examine whether Ca2+ released by PF pulses triggered from different sub-cellular compartments (SR, ER, mitochondria) underlies the inhibitory effect of PF on the channel current, pharmacological agents and ionic substitutions were employed to probe this possibility. No significant difference in effectiveness of PF pulses to suppress ICa or IBa (used to inhibit CICR) was found between control cells and those exposed to U73122 and 2-APB (PLC and IP3R pathway modulators), thapsigargin and BAPTA (SERCA2a modulator), dinitrophenol, FCCP and Ru360 (mitochondrial inhibitors), l-NAME (NOS inhibitor signaling), cAMP and Pertussis toxin (Gi protein modulator). We concluded that the rapid and reversible modulation of the Ca2+ channel by PF pulses is independent of intracellular release of Ca2+ and Ca2+ dependent inactivation of the channel and may represent direct mechanical regulatory effect on the channel protein in addition to previously reported Ca2+-release or entry dependent mechanism.

A Molecular Switch Driving Inactivation in the Cardiac K+ Channel hERG

K+ channels control transmembrane action potentials by gating open or closed in response to external stimuli. Inactivation gating, involving a conformational change at the K+ selectivity filter, has recently been recognized as a major K+ channel regulatory mechanism. In the K+ channel hERG, inactivation controls the length of the human cardiac action potential. Mutations impairing hERG inactivation cause life-threatening cardiac arrhythmia, which also occur as undesired side effects of drugs. In this paper, we report atomistic molecular dynamics simulations, complemented by mutational and electrophysiological studies, which suggest that the selectivity filter adopts a collapsed conformation in the inactivated state of hERG. The selectivity filter is gated by an intricate hydrogen bond network around residues S620 and N629. Mutations of this hydrogen bond network are shown to cause inactivation deficiency in electrophysiological measurements. In addition, drug-related conformational changes around the central cavity and pore helix provide a functional mechanism for newly discovered hERG activators.

La mucoviscidose et autres canalopathies

Mutations in cystic fibrosis transmembrane conductance regulator gene, CFTR, are responsible for cystic fibrosis, CF, a channelopathie. CFTR protein is a multifunctional protein with a main function of Cl(-) channel. CFTR is expressed in epithelia (upper airways, intestine, pancreas etc.). In the first part of this revue, we describe the main properties of CFTR underlying that it is not only a Cl(-) channel protein but also a multifunctional protein. We present a hypothesis which postulates that CFTR is a hub protein interacting with more than 140 proteins, and through these interactions regulates a number of functions which are abnormal in CF (ion transport, inflammation etc.). In the second part of the revue we briefly present a selection of other epithelial channelopathies due to mutations in genes of other Cl(-) or cation channels. Of note, these channels either interacts with CFTR or are considered as alternative channels in CF, and, as such, are targets for pharmacotherapies. We want to leave the reader with a message that to investigate channalopathies, to dissect the molecular mechanisms underlying channels'activity, allow not only to better understand basic mechanisms of channel regulation but in fine, to propose new targets for pharmacotherapies.

A remarkably stable TipE gene cluster: evolution of insect Para sodium channel auxiliary subunits.

BackgroundFirst identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV) channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures.ResultsWe identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa) channels, which suggests that ion channel regulatory partners have evolved distinct lineage-specific characteristics.ConclusionsTipE-like genes form a remarkably conserved genomic cluster across all examined insect genomes. This study reveals likely structural and functional constraints on the genomic evolution of insect TipE gene family members maintained in synteny over hundreds of millions of years of evolution. The likely common origin of these NaV channel regulators with BKCa auxiliary subunits highlights the evolutionary plasticity of ion channel regulatory mechanisms.

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement

Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells. In our previous study, the effect of methyl-beta-cyclodextrin (MbCD), cholesterol-sequestering agent, on Ca 2+-permeable stretch-activated cation channels (SACs) has been described. Here, cell-attached patch-clamp method was employed to search for the mechanisms of cholesterol-dependent regulation of SACs and to clarify functional contribution of lipid bilayer and submembranous cytoskeleton to channel gating. Cholesterol-depleting treatment with MbCD significantly decreased open probability of SACs whereas alpha-cyclodextrin had no effect. F-actin disassembly fully restored high level of SAC activity in cholesterol-depleted cells. Particularly, treatment with cytochalasin D or latrunculin B abrogated inhibitory effect of MbCD on stretch-activated currents. Single channel analysis and fluorescent imaging methods indicate that inhibition of SACs after cholesterol depletion is mediated via actin remodeling initiated by disruption of lipid rafts. Our data reveal a novel mechanism of channel regulation by membrane cholesterol and lipid rafts.

Dantrolene, a Therapeutic Agent for Malignant Hyperthermia, Markedly Improves the Function of Failing Cardiomyocytes by Stabilizing Interdomain Interactions Within the Ryanodine Receptor

We sought to investigate the effect of dantrolene, a drug generally used to treat malignant hyperthermia, on the Ca2+ release and cardiomyocyte function in failing hearts. The N-terminal (N: 1-600) and central (C: 2000-2500) domains of the ryanodine receptor (RyR) harbor many mutations associated with malignant hyperthermia in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that interdomain interaction between these regions plays an important role in the mechanism of channel regulation. Sarcoplasmic reticulum vesicles and cardiomyocytes were isolated from the left ventricular muscles of dogs (normal or rapid ventricular pacing for 4 weeks), for Ca2+ leak, transient, and spark assays. To assess the zipped or unzipped state of the interacting domains, the RyR was labeled fluorescently with methylcoumarin acetate in a site-directed manner. We used a quartz-crystal microbalance technique to identify the dantrolene binding site within the RyR2. Dantrolene specifically bound to domain 601-620 in RyR2. In the sarcoplasmic reticulum isolated from pacing-induced failing dog hearts, the defective interdomain interaction (domain unzipping) had already occurred, causing spontaneous Ca2+ leak. Dantrolene suppressed both domain unzipping and the Ca2+ leak, demonstrating identical drug concentration-dependence (IC50 = 0.3 micromol/l). In failing cardiomyocytes, both diastolic Ca2+ sparks and delayed afterdepolarization were observed frequently, but 1 micromol/l dantrolene inhibited both events. Dantrolene corrects defective interdomain interactions within RyR2 in failing hearts, inhibits spontaneous Ca2+ leak, and in turn improves cardiomyocyte function in failing hearts. Thus, dantrolene may have a potential to treat heart failure, specifically targeting the RyR2.

Sustainable TRPM4 Channel Activity in Freshly‐Isolated Cerebral Artery Smooth Muscle Cells

Localized control of blood flow and arterial constriction in response to changes in intraluminal pressure are dependent on the intrinsic regulation of smooth muscle cell membrane potential. The melastatin transient receptor potential (TRP) channel TRPM4 is highly selective for monovalent cations, is activated by elevated levels of intracellular Ca2+, and is a critical regulator of smooth muscle membrane depolarization, which serves to open voltage dependent Ca2+channels (VDCC), and resulting in myocyte contraction. Interestingly, Ca2+ activates and inactivates TRPM4, causing the channel to rapidly inactivate under conventional whole‐cell patch clamp conditions. When the slow Ca2+ buffer, ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA), was included in the pipette solution, we identified a transient tetraethylammonium (TEA)‐insensitive inward Na+ current in freshly isolated cerebral arterial myocytes. siRNA‐mediated silencing of TRPM4 and the recently described TRPM4 inhibitor, 9‐phenanthrol, both diminish the open probability (NPo) compared to controls, strongly suggesting the molecular identity of the channel to be TRPM4. This study demonstrates a practical method to record sustained TRPM4 activity and allows for further investigation into mechanisms of channel regulation. AHA0535226N; F31HL094145‐01.

Elongation Factor 3, EF3, Associates with the Calcium Channel Cch1 and Targets Cch1 to the Plasma Membrane inCryptococcus neoformans

Ca2+-mediated signaling events in eukaryotic cells are initiated by Ca2+ channels located in the plasma membranes and endomembranes. Cch1, a high-affinity Ca2+ channel in the plasma membranes of Cryptococcus neoformans and other fungi, plays a role in many different cellular processes, but the mechanisms that regulate Cch1 are not well understood. A Ras recruitment two-hybrid screen was used to identify protein partners of Cch1 as a means of identifying possible mechanisms of channel regulation. Here, we show that Cch1 specifically associates with a cytoplasmic protein known as elongation factor 3 (EF3). The robust interaction between the cytosolic C terminus of the Cch1 protein and EF3 shown here was confirmed by demonstrating that Cch1 could coimmunoprecipitate with EF3 in yeast lysates. To examine the effects of EF3 on Cch1 behavior, we altered the EF3 gene function by constructing a C. neoformans antisense EF3 repression strain. Our results show that the repression of EF3 led to the mislocalization of Cch1, suggesting a role for EF3 in targeting Cch1 to the plasma membrane of C. neoformans. Consistent with this notion, the antisense EF3 repression strain displayed a growth defect under conditions of limited extracellular Ca2+. Collectively, these results suggest that EF3 and Cch1 are functionally coupled and that EF3 has a function apart from its role in the protein translation cycle.

Milk Marketing Policy Options for the Dairy Industry in New England

New England dairy farmers are under intense price pressure resulting from important growth in milk production from lower cost of production Southwest states as well as by retailers’ market power. Agricultural officials and legislative bodies in New England and in other Northeast US states are aware of these pressures and have been reacting with emergency dairy farm aid, following a very low 2006 milk price, and with state legislations in an attempt to address perceived excess retailing margins for fluid milk. In this paper, we suggest that a sigmoid demand relationship exists for fluid milk. This demand relationship would explain fluid milk asymmetric price transmission, high-low pricing, and the creation of a large retailing margin (chain surplus) often observed for fluid milk. It is also argued that a sigmoid demand relationship offers an opportunity for state legislators to help Northeast dairy farmers capturing a larger share of the dollar of the consumers through various policy options. Therefore, 5 milk market channel regulatory mechanisms (status quo, price gouging, supply control, fair share policy, and chain surplus return) are discussed and compared. The supply control mechanism was found the most effective at redistributing the chain surplus, associated with the sigmoid demand relationship for fluid milk, to dairy farmers. However, this option is unlikely to be politically acceptable in the United States. Second-best options for increasing dairy farmers’ share of the consumers’ dollar are the fair price policy and the chain surplus return. The former mechanism would distribute the chain surplus between retailers, processors, and farmers, whereas the latter would distribute it between consumers, retailers, and farmers. Remaining mechanisms would either transfer the chain surplus to retailers (status quo) or to consumers (price gouging).

Rapid ligand‐regulated gating kinetics of single inositol 1,4,5‐trisphosphate receptor Ca 2+ release channels

The ubiquitous inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel is engaged by thousands of plasma membrane receptors to generate Ca(2+) signals in all cells. Understanding how complex Ca(2+) signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP(3) and Ca(2+), which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP(3)R channels in native endoplasmic reticulum membrane to rapid ligand concentration changes with millisecond resolution, using a new patch-clamp configuration. The kinetics of channel activation and deactivation showed novel Ca(2+) regulation and unexpected ligand cooperativity. The kinetics of Ca(2+)-mediated channel inhibition showed the single-channel bases for fundamental Ca(2+) release events and Ca(2+) release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca(2+) signals.

Phospholipid Minds the Gap (Junction)

Gap junction channels allow communication in the form of small molecules that flow between adjacent cells. Conductance through gap junctions formed of the connexin43 (Cx43) protein is decreased after stimulation of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) at the cell surface, but the actual mechanism of channel regulation has been unclear. van Zeijl et al . report that in Rat-1 fibroblasts, one way gap junction conductance can be regulated is through changes in the abundance of the phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]. The authors confirmed decreased conductance of gap junctions formed from Cx43 in cells transfected with an active form of the G protein alpha subunit Gα q . Gα q activates phospholipase C-β (PLC-β), which catalyzes hydrolysis of PtdIns(4,5)P 2 , forming the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol, and experiments with a fluorescently labeled probe containing a pleckstrin homology domain that binds PtdIns(4,5)P 2 revealed that PtdIns(4,5)P 2 was depleted from the plasma membrane in cells expressing the active Gα q protein. Depletion of PLC-β3 with RNAi prevented depletion of PtdIns(4,5)P 2 from the plasma membrane in cells treated with GPCR agonists. The communication with the gap junction appeared to require PtdIns(4,5)P 2 itself, rather than IP 3 or DAG, and channel inhibition could be reproduced by depletion of PtdIns(4,5)P 2 with an engineered version of phosphoinositide 5-phosphatase targeted to the plasma membrane. Similarly, overexpression of PtdIns(4)P 5-kinase [to maintain the abundance of PtdIns(4,5)P 2 at the membrane] also prevented receptor-mediated decreases in gap junctional communication. Immunoprecipitation experiments showed that PLC-β3 associated with the scaffold protein ZO-1 (zona occludens 1), and depletion of ZO-1 with RNAi also reduced receptor-mediated regulation of gap junction channels. Thus, the authors propose that ZO-1 helps assemble complexes containing PLC-β3 and Cx43. It’s not altered second messenger production that shuts down the channel but rather the action of PLC-β3 to deplete membrane concentrations of PtdIns(4,5)P 2 , which appears to directly modulate conductance of gap junctions as it does other ion channels and transporters. L. van Zeijl, B. Ponsioen, B. N. G. Giepmans, A. Ariaens, F. R. Postma , P. Várnai, T. Balla, N. Divecha, K. Jalink, W. H. Moolenaar, Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate. J. Cell Biol. 177 , 881-891 (2007). [Abstract] [Full Text]

Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C‐terminal domain

Voltage-gated Ca(2+) channels of the Ca(V)1 family initiate excitation-contraction coupling in cardiac, smooth, and skeletal muscle and are primary targets for regulation by the sympathetic nervous system in the 'fight-or-flight' response. In the heart, activation of beta-adrenergic receptors greatly increases the L-type Ca(2+) current through Ca(V)1.2 channels, which requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15). Surprisingly, the site of interaction of PKA and AKAP15 lies in the distal C-terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the proteolytically cleaved distal C-terminal domain forms a specific molecular complex with the truncated alpha(1) subunit and serves as a potent autoinhibitory domain. Formation of the autoinhibitory complex greatly reduces the coupling efficiency of voltage sensing to channel opening and shifts the voltage dependence of activation to more positive membrane potentials. Ab initio structural modelling and site-directed mutagenesis revealed a binding interaction between a pair of arginine residues in a predicted alpha-helix in the proximal C-terminal domain and a set of three negatively charged amino acid residues in a predicted helix-loop-helix bundle in the distal C-terminal domain. Disruption of this interaction by mutation abolished the inhibitory effects of the distal C-terminus on Ca(V)1.2 channel function. These results provide the first functional characterization of this autoinhibitory complex, which may be a major form of the Ca(V)1 family Ca(2+) channels in cardiac and skeletal muscle cells, and reveal a unique ion channel regulatory mechanism in which proteolytic processing produces a more effective autoinhibitor of Ca(V)1.2 channel function.

Reactions with Dye Free Radicals Reveal Weak Redox Properties of Drugs

The calcium release channel (CRC) of the skeletal sarcoplasmic reticulum is rich in thiol groups and is strongly regulated by covalent modification of these thiols. Oxidizing reagents activate the release channel, whereas reducing reagents inhibit the channel. However, most CRC regulators are not thiol reagents. Here, we propose that reversible redox interactions are involved in regulation of the CRC by nonthiol reagents. This hypothesis was tested with several CRC regulators. The local anesthetics tetracaine, procaine and QX-314, which block the CRC, behaved as electron donors in reactions with dye free radicals. In contrast, ryanodine, caffeine, doxorubicin and daunorubicin, compounds known to activate the release channel, all accepted electrons from dye anion radicals. Moreover, release of Ca2+ from SR initiated by doxorubicin (acceptor) was antagonized by local anesthetics (donors). Based on the redox characterization of CRC modulators, we propose a functional model in which channel inhibitors and activators act as weak electron donors and acceptors, respectively, and shift the thiol-disulfide balance within the release protein. The consequence of this model is that, in spite of the chemical diversity of CRC modulators, a common mechanism of channel regulation involves the transient exchange of electrons between the activator/inhibitor and the CRC.

Regulation of I(Cl,swell) in neuroblastoma cells by G protein signaling pathways.

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) activated the I(Cl,swell) anion channel in N1E115 neuroblastoma cells in a swelling-independent manner. GTPgammaS-induced current was unaffected by ATP removal and broadly selective tyrosine kinase inhibitors, demonstrating that phosphorylation events do not regulate G protein-dependent channel activation. Pertussis toxin had no effect on GTPgammaS-induced current. However, cholera toxin inhibited the current approximately 70%. Exposure of cells to 8-bromoadenosine 3',5'-cyclic monophosphate did not mimic the effect of cholera toxin, and its inhibitory action was not prevented by treatment of cells with an inhibitor of adenylyl cyclase. These results demonstrate that GTPgammaS does not act through Galpha(i/o) GTPases and that Galpha(s)/Gbetagamma G proteins inhibit the channel and/or channel regulatory mechanisms through cAMP-independent mechanisms. Swelling-induced activation of I(Cl,swell) was stimulated two- to threefold by GTPgammaS and inhibited by 10 mM guanosine 5'-O-(2-thiodiphosphate). The Rho GTPase inhibitor Clostridium difficile toxin B inhibited both GTPgammaS- and swelling-induced activation of I(Cl,swell). Taken together, these findings indicate that Rho GTPase signaling pathways regulate the I(Cl,swell) channel via phosphorylation-independent mechanisms.