Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

Targeted ubiquitination of sensory neuron calcium channels reduces the development of neuropathic pain.

Neuropathic pain caused by lesions to somatosensory neurons due to injury or disease is a widespread public health problem that is inadequately managed by small-molecule therapeutics due to incomplete pain relief and devastating side effects. Genetically encoded molecules capable of interrupting nociception have the potential to confer long-lasting analgesia with minimal off-target effects. Here, we utilize a targeted ubiquitination approach to achieve a unique posttranslational functional knockdown of high-voltage-activated calcium channels (HVACCs) that are obligatory for neurotransmission in dorsal root ganglion (DRG) neurons. CaV-aßlator comprises a nanobody targeted to CaV channel cytosolic auxiliary ß subunits fused to the catalytic HECT domain of the Nedd4-2 E3 ubiquitin ligase. Subcutaneous injection of adeno-associated virus serotype 9 encoding CaV-aßlator in the hind paw of mice resulted in the expression of the protein in a subset of DRG neurons that displayed a concomitant ablation of CaV currents and also led to an increase in the frequency of spontaneous inhibitory postsynaptic currents in the dorsal horn of the spinal cord. Mice subjected to spare nerve injury displayed a characteristic long-lasting mechanical, thermal, and cold hyperalgesia underlain by a dramatic increase in coordinated phasic firing of DRG neurons as reported by in vivo Ca2+ spike recordings. CaV-aßlator significantly dampened the integrated Ca2+ spike activity and the hyperalgesia in response to nerve injury. The results advance the principle of targeting HVACCs as a gene therapy for neuropathic pain and demonstrate the therapeutic potential of posttranslational functional knockdown of ion channels achieved by exploiting the ubiquitin-proteasome system.

Targeted deubiquitination rescues distinct trafficking-deficient ion channelopathies.

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density, but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model, enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common (ΔF508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether, targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking, but also introduces a new tool for deconstructing the ubiquitin code in situ.

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

RATIONALE: Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. OBJECTIVE: The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during ß-adrenergic stimulation, and in heart failure. METHODS AND RESULTS: We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and ß-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVß to α1C I-II loop and eliminated ß-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to ß-adrenergic agonists when reconstituted heterologously with ß2B and Rad or transgenically expressed in cardiomyocytes. CONCLUSIONS: Ca2+ channel activity is dynamically modulated under basal conditions, during ß-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVß binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the ß-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by ß-adrenergic agonists/PKA also requires this rigid linker and ß-binding to α1C.

Design and Applications of Genetically-Encoded Voltage-Dependent Calcium Channel Inhibitors.

Ca2+ influx through high-voltage-gated Ca2+ channels (HVGCCs; CaV1/CaV2) is an exceptionally powerful and versatile signal that controls numerous cell and physiological functions including neurotransmission, muscle contraction, and regulation of gene expression. The impressive ability of a singular signal, Ca2+ influx, to have such a plethora of functional outcomes is enabled by: molecular diversity of HVGCC pore-forming α1 and auxiliary subunits; organization of HVGCCs with extrinsic modulatory and effector protein to form discrete macromolecular complexes with unique properties; distinctive distribution of HVGCCs into separate subcellular compartments; and varying expression profiles of HVGCC isoforms among different tissues and organs. The capacity to block HVGCCs with selectivity and specificity with respect to the different levels of their organization is critical for fully understanding the scope of functional consequences of Ca2+ influx through them, and is also important for realizing their full potential as therapeutic targets. In this review, we discuss the gaps in the current landscape of small-molecule HVGCC blockers and how these may be addressed with designer genetically-encoded Ca2+ channel inhibitors (GECCIs) that draw inspiration from physiological protein inhibitors of HVGCCs.

Controlling ion channel function with renewable recombinant antibodies.

Selective ion channel modulators play a critical role in physiology in defining the contribution of specific ion channels to physiological function and as proof of concept for novel therapeutic strategies. Antibodies are valuable research tools that have broad uses including defining the expression and localization of ion channels in native tissue, and capturing ion channel proteins for subsequent analyses. In this review, we detail how renewable and recombinant antibodies can be used to control ion channel function. We describe the different forms of renewable and recombinant antibodies that have been used and the mechanisms by which they modulate ion channel function. We highlight the use of recombinant antibodies that are expressed intracellularly (intrabodies) as genetically encoded tools to control ion channel function. We also offer perspectives of avenues of future research that may be opened by the application of emerging technologies for engineering recombinant antibodies for enhanced utility in ion channel research. Overall, this review provides insights that may help stimulate and guide interested researchers to develop and incorporate renewable and recombinant antibodies as valuable tools to control ion channel function.

A light-gated potassium channel for sustained neuronal inhibition.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVß-subunits. To investigate latent CaVß-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) ß2a or a mutant version (ß2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT ß2a By contrast, when channels were reconstituted with ß2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVß, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent ß-binding-independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaV channel blockers.

Designer genetically encoded voltage-dependent calcium channel inhibitors inspired by RGK GTPases.

High-voltage-activated calcium (CaV 1/CaV 2) channels translate action potentials into Ca2+ influx in excitable cells to control essential biological processes that include; muscle contraction, synaptic transmission, hormone secretion and activity-dependent regulation of gene expression. Modulation of CaV 1/CaV 2 channel activity is a powerful mechanism to regulate physiology, and there are a host of intracellular signalling molecules that tune different aspects of CaV channel trafficking and gating for this purpose. Beyond normal physiological regulation, the diverse CaV channel modulatory mechanisms may potentially be co-opted or interfered with for therapeutic benefits. CaV 1/CaV 2 channels are potently inhibited by a four-member sub-family of Ras-like GTPases known as RGK (Rad, Rem, Rem2, Gem/Kir) proteins. Understanding the mechanisms by which RGK proteins inhibit CaV 1/CaV 2 channels has led to the development of novel genetically encoded CaV channel blockers with unique properties; including, chemo- and optogenetic control of channel activity, and blocking channels either on the basis of their subcellular localization or by targeting an auxiliary subunit. These genetically encoded CaV channel inhibitors have outstanding utility as enabling research tools and potential therapeutics.

Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

Calcium influx through the voltage-dependent L-type calcium channel (CaV1.2) rapidly increases in the heart during "fight or flight" through activation of the ß-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of ß-adrenergic activation of cardiac CaV1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α1C and C-terminal proteolytic cleavage of the α1C subunit. We generated transgenic mice expressing an α1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for ß-adrenergic regulation of CaV1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute ß-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α1C Prevention of C-terminal cleavage did not alter ß-adrenergic stimulation of CaV1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α1C in ß-adrenergic stimulation of CaV1.2, and show that phosphoregulatory sites on α1C are not redundant and do not each fractionally contribute to the net stimulatory effect of ß-adrenergic stimulation. Further, proteolytic cleavage of α1C is not required for ß-adrenergic stimulation of CaV1.2.

Regulation of Blood Pressure by Targeting CaV1.2-Galectin-1 Protein Interaction.

BACKGROUND: L-type CaV1.2 channels play crucial roles in the regulation of blood pressure. Galectin-1 (Gal-1) has been reported to bind to the I-II loop of CaV1.2 channels to reduce their current density. However, the mechanistic understanding for the downregulation of CaV1.2 channels by Gal-1 and whether Gal-1 plays a direct role in blood pressure regulation remain unclear. METHODS: In vitro experiments involving coimmunoprecipitation, Western blot, patch-clamp recordings, immunohistochemistry, and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 downregulates CaV1.2 channel in transfected, human embryonic kidney 293 cells, smooth muscle cells, arteries from Lgasl1-/- mice, rat, and human patients. In vivo experiments involving the delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting CaV1.2-Gal-1 interaction on blood pressure monitored by tail-cuff or telemetry methods. RESULTS: Our study reveals that Gal-1 is a key regulator for proteasomal degradation of CaV1.2 channels. Gal-1 competed allosterically with the CaVß subunit for binding to the I-II loop of the CaV1.2 channel. This competitive disruption of CaVß binding led to CaV1.2 degradation by exposing the channels to polyubiquitination. It is notable that we demonstrated that the inverse relationship of reduced Gal-1 and increased CaV1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice because of the upregulated CaV1.2 protein level in arteries. To directly regulate blood pressure by targeting the CaV1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1 by a miniosmotic pump, and this specific disruption of CaV1.2-Gal-1 coupling increased smooth muscle CaV1.2 currents, induced larger arterial contraction, and caused hypertension in rats. In contrasting experiments, overexpression of Gal-1 in smooth muscle by a single bolus of AAV5-Gal-1 significantly reduced blood pressure in spontaneously hypertensive rats. CONCLUSIONS: We have defined molecularly that Gal-1 promotes CaV1.2 degradation by replacing CaVß and thereby exposing specific lysines for polyubiquitination and by masking I-II loop endoplasmic reticulum export signals. This mechanistic understanding provided the basis for targeting CaV1.2-Gal-1 interaction to demonstrate clearly the modulatory role that Gal-1 plays in regulating blood pressure, and offering a potential approach for therapeutic management of hypertension.

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.

Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels.

Ca(2+)-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca(2+) is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach--channel inactivation induced by membrane-tethering of an associated protein (ChIMP)--we discovered that Ca(2+)-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca(2+)-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM1234. These effects are Ca(2+)-dependent sensitization of activation (CDSA) and Ca(2+)-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca(2+)-activated chloride channels.

High-fat diet-dependent modulation of the delayed rectifier K(+) current in adult guinea pig atrial myocytes.

Obesity is associated with hyperlipidemia, electrical remodeling of the heart, and increased risk of supraventricular arrhythmias in both male and female patients. The delayed rectifier K(+) current (IK), is an important regulator of atrial repolarization. There is a paucity of studies on the functional role of IK in response to obesity. Here, we assessed the obesity-mediated functional modulation of IK in low-fat diet (LFD), and high-fat diet (HFD) fed adult guinea pigs. Guinea pigs were randomly divided into control and obese groups fed, ad libitum, with a LFD (10 kcal% fat) or a HFD (45 kcal% fat) respectively. Action potential duration (APD), and IK were studied in atrial myocytes and IKr and IKs in HEK293 cells using whole-cell patch clamp electrophysiology. HFD guinea pigs displayed a significant increase in body weight, total cholesterol and total triglycerides within 50 days. Atrial APD at 30% (APD30) and 90% (APD90) repolarization were shorter, while atrial IK density was significantly increased in HFD guinea pigs. Exposure to palmitic acid (PA) increased heterologously expressed IKr and IKs densities, while oleic acid (OA), severely reduced IKr and had no effect on IKs. The data are first to show that in obese guinea pigs abbreviated APD is due to increased IK density likely through elevations of PA. Our findings may have crucial implications for targeted treatment options for obesity-related arrhythmias.

Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing.

Manipulating expression of large genes (>6 kb) in adult cardiomyocytes is challenging because these cells are only efficiently transduced by viral vectors with a 4-7 kb packaging capacity. This limitation impedes understanding structure-function mechanisms of important proteins in heart. L-type calcium channels (LTCCs) regulate diverse facets of cardiac physiology including excitation-contraction coupling, excitability, and gene expression. Many important questions about how LTCCs mediate such multidimensional signaling are best resolved by manipulating expression of the 6.6 kb pore-forming α1C-subunit in adult cardiomyocytes. Here, we use split-intein-mediated protein transsplicing to reconstitute LTCC α1C-subunit from two distinct halves, overcoming the difficulty of expressing full-length α1C in cardiomyocytes. Split-intein-tagged α1C fragments encoding dihydropyridine-resistant channels were incorporated into adenovirus and reconstituted in cardiomyocytes. Similar to endogenous LTCCs, recombinant channels targeted to dyads, triggered Ca(2+) transients, associated with caveolin-3, and supported ß-adrenergic regulation of excitation-contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes.

Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation.

Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (Ca(V)1.2 currents, Ca(2+) transients, Ca(V)ß binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized Rad(S105N) and Rem(T94N), which are the cognate mutants to Ras(S17N), a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either Rad(S105N) or Rem(T94N) strongly inhibited reconstituted Ca(V)1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that Rad(S105N) is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or Rad(S105N) in cardiomyocytes dramatically blocked L-type calcium current (I(Ca,L)) and inhibited Ca(2+)-induced Ca(2+) release, contradicting reports that Rad(S105N) acts as a dominant negative in heart. By contrast, Rem(T94N) was significantly less effective than wt Rem at inhibiting I(Ca,L) and Ca(2+) transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both Rad(S105N) and Rem(T94N) had moderately reduced binding affinity for Ca(V)ßs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in Ca(V)1.2 regulation.

Regulation of voltage-dependent calcium channels by RGK proteins.

RGK proteins belong to the Ras superfamily of monomeric G-proteins, and currently include four members - Rad, Rem, Rem2, and Gem/Kir. RGK proteins are broadly expressed, and are the most potent known intracellular inhibitors of high-voltage-activated Ca²âº (Ca(V)1 and Ca(V)2) channels. Here, we review and discuss the evidence in the literature regarding the functional mechanisms, structural determinants, physiological role, and potential practical applications of RGK-mediated inhibition of Ca(V)1/Ca(V)2 channels. This article is part of a Special Issue entitled: Calcium channels.