RGK Proteins Research Articles

The RGK protein rrad regulates cardiac calcium levels in zebrafish

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain Background/Introduction High voltage-activated Ca2+ (CaV) channels are essential for excitation-contraction coupling. RGK proteins (Gem, Rad, Rem, and Rem2) are small Ras-like G proteins that bind CaV β-subunits and profoundly inhibit CaV1/CaV2 channels. A recent study showed that Rad inhibits CaV1.2 channels in cardiomyocytes, thus mediating the enhanced inotropy and chronotropy by β-adrenergic agonists during the fight-or-flight response. The zebrafish has emerged as a useful in vivo animal model in cardiovascular physiology and pathophysiology. The role of zebrafish rrad, the orthologe of the human RAD, in the regulation of cardiac Ca2+ homeostasis in zebrafish has not been investigated. Purpose The aim of this study was to investigate the role of rrad in cardiac Ca2+ regulation in zebrafish. Methods We evaluated the cardiac Ca2+ dynamics, heart rhythm, and hemodynamics in transgenic zebrafish larvae that overexpress rrad in the heart, as well as in heterozygous rrad knockout larvae. Measurements were done in the beating heart of 3 dpf transgenic zebrafish larvae expressing the fluorescent Ca2+ biosensor Twitch-4. Results In some larvae, overexpression of rrad induced a severe enlargement of the atrium with pericardial edema and a high mortality rate. In addition, the ventricle displayed low Ca2+ levels and reduced contractility. The remaining larvae had no altered morphology, but the ventricular Ca2+ levels were reduced. On the other hand, preliminary results showed that down regulation of rrad in zebrafish larvae increased atrial and ventricular Ca2+ levels, like what was found in Rad knockout mice. Conclusion Taken together, these findings suggest that rrad in the zebrafish heart, as in mammals, is an inhibitor of the Ca2+ entry, presumably through CaV1/CaV2 channels, and demonstrate the zebrafish's utility as a model to study the involvement of rrad in cardiac function.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

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

High‐voltage‐activated calcium (CaV1/CaV2) 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 CaV1/CaV2 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. CaV1/CaV2 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 CaV1/CaV2 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.

Autism-associated mutations in the CaVβ2 calcium-channel subunit increase Ba2+-currents and lead to differential modulation by the RGK-protein Gem

Voltage-gated calcium-channels (VGCCs) are heteromers consisting of several subunits. Mutations in the genes coding for VGCC subunits have been reported to be associated with autism spectrum disorder (ASD). In a previous study, we identified electrophysiologically relevant missense mutations of CaVβ2 subunits of VGCCs. From this, we derived the hypothesis that several CaVβ2-mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a CaVβ2d backbone, we performed extensive whole-cell and single-channel patch-clamp analyses of Ba2+ currents carried by Cav1.2 pore subunits co-transfected with the previously described CaVβ2 mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated CaVβ2d subunits with the RGK protein Gem was analyzed by co-immunoprecipitation assays and electrophysiological studies. Patch-clamp analyses revealed that all mutations increase Ba2+ currents, e.g. by decreasing inactivation or increasing fraction of active sweeps. All CaVβ2 mutations interact with Gem, but differ in the extent and characteristics of modulation by this RGK protein (e.g. decrease of fraction of active sweeps: CaVβ2d_G167S > CaVβ2d_V2D > CaVβ2d_S197F). In conclusion, patch-clamp recordings of ASD-associated CaVβ2d mutations revealed differential modulation of Ba2+ currents carried by CaV1.2 suggesting kind of an “electrophysiological fingerprint” each. The increase in current finally observed with all CaVβ2d mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism.

RGK Proteins Preferentially Inhibit Fast-Inactivating Voltage-Gated Calcium Channels: Implications for Human Disease

Voltage-gated calcium channels (VGCC) are critical for nerve, heart, and skeletal muscle function, and their mutations can cause neurological and cardiovascular disease. Some of these mutations alter channel inactivation, leading to aberrant Ca2+ influx into cells. For example, Timothy syndrome (TS) is characterized by severe cardiomyopathy, and is often accompanied by autism spectrum disorder. The abnormalities arise from a point mutation in the L-type VGCC that slows channel inactivation. On the other hand, some types of migraine and epilepsy are associated with point mutations that speed the inactivation of P/Q-type VGCC. RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that strongly inhibit L-, N-, P/Q- and R-type VGCC. They are difficult to study because their overexpression nearly completely abolishes VGCC currents. Thus, we studied RGK-mediated inhibition using two-electrode voltage clamp (TEVC) in Xenopus oocytes, where we titrated the amount of injected RNA to achieve a more physiological level of VGCC inhibition of ∼50%. Under these conditions, we discovered that mutations that speed inactivation, such as those that cause familial hemiplegic migraine, turn channels hypersensitive to Gem inhibition. Remarkably, when we challenged the non-inactivating TS channel with Gem, we found that it was insensitive to inhibition. Furthermore, expressing VGCC with the β2a subunit, a Ca2+ channel auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition.This could potentially occur if Gem were to trap channels in the inactivated state, thereby inhibiting inactivating channels, while sparing non-inactivating channels. Our additional findings corroborate this since channel recovery from inactivation is much slower in the presence of Gem.These results shed light on a new type of VGCC regulation. In addition, our results point to RGKs as potential players in calcium channelopathies.

Molecular mechanisms and physiological relevance of RGK proteins in the heart.

The primary route of Ca2+ entry into cardiac myocytes is via 1,4-dihydropyridine-sensitive, voltage-gated L-type Ca2+ channels. Ca2+ influx through these channels influences duration of action potential and engages excitation-contraction (EC) coupling in both the atria and the myocardium. Members of the RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTP-binding proteins are potent, endogenously expressed inhibitors of cardiac L-type channels. Although much work has focused on the molecular mechanisms by which RGK proteins inhibit the CaV 1.2 and CaV 1.3 L-type channel isoforms that expressed in the heart, their impact on greater cardiac function is only beginning to come into focus. In this review, we summarize recent findings regarding the influence of RGK proteins on normal cardiac physiology and the pathological consequences of aberrant RGK activity.

Similar molecular determinants on Rem mediate two distinct modes of inhibition of CaV1.2 channels

ABSTRACTRad/Rem/Rem2/Gem (RGK) proteins are Ras-like GTPases that potently inhibit all high-voltage-gated calcium (CaV1/CaV2) channels and are, thus, well-positioned to tune diverse physiological processes. Understanding how RGK proteins inhibit CaV channels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded CaV channel blockers. We previously reported that Rem can block surface CaV1.2 channels in 2 independent ways that engage distinct components of the channel complex: (1) by binding auxiliary β subunits (β-binding-dependent inhibition, or BBD); and (2) by binding the pore-forming α1C subunit N-terminus (α1C-binding-dependent inhibition, or ABD). By contrast, Gem uses only the BBD mechanism to block CaV1.2. Rem molecular determinants required for BBD CaV1.2 inhibition are the distal C-terminus and the guanine nucleotide binding G-domain which interact with the plasma membrane and CaVβ, respectively. However, Rem determinants for ABD CaV1.2 inhibition are unknown. Here, combining fluorescence resonance energy transfer, electrophysiology, systematic truncations, and Rem/Gem chimeras we found that the same Rem distal C-terminus and G-domain also mediate ABD CaV1.2 inhibition, but with different interaction partners. Rem distal C-terminus interacts with α1C N-terminus to anchor the G-domain which likely interacts with an as-yet-unidentified site. In contrast to some previous studies, neither the C-terminus of Rem nor Gem was sufficient to inhibit CaV1/CaV2 channels. The results reveal that similar molecular determinants on Rem are repurposed to initiate 2 independent mechanisms of CaV1.2 inhibition.

Rem2, a member of the RGK family of small GTPases, is enriched in nuclei of the basal ganglia.

Rem2 is a member of the RGK subfamily of RAS small GTPases. Rem2 inhibits high voltage activated calcium channels, is involved in synaptogenesis, and regulates dendritic morphology. Rem2 is the primary RGK protein expressed in the nervous system, but to date, the precise expression patterns of this protein are unknown. In this study, we characterized Rem2 expression in the mouse nervous system. In the CNS, Rem2 mRNA was detected in all regions examined, but was enriched in the striatum. An antibody specific for Rem2 was validated using a Rem2 knockout mouse model and used to show abundant expression in striatonigral and striatopallidal medium spiny neurons but not in several interneuron populations. In the PNS, Rem2 was abundant in a subpopulation of neurons in the trigeminal and dorsal root ganglia, but was absent in sympathetic neurons of superior cervical ganglia. Under basal conditions, Rem2 was subject to post-translational phosphorylation, likely at multiple residues. Further, Rem2 mRNA and protein expression peaked at postnatal week two, which corresponds to the period of robust neuronal maturation in rodents. This study will be useful for elucidating the functions of Rem2 in basal ganglia physiology.

Inhibition of Voltage-Gated Calcium Channels by RGK Proteins.

Due to their essential biological roles, voltage-gated calcium channels (VGCCs) are regulated by a myriad of molecules and mechanisms. Fifteen years ago, RGK proteins were discovered to bind the VGCC β subunit (Cavβ) and potently inhibit high-voltage activated Ca(2+) channels. RGKs (Rad, Rem, Rem2 and Gem/Kir) are a family of monomeric small GTPases belonging to the superfamily of Ras GTPases. They exert dual inhibitory effects on VGCCs, decreasing surface expression and suppressing surface channels through immobilization of the voltage sensor or reduction of channel open probability. While Cavβ is required for all forms of RGK inhibition, not all inhibition is mediated by the RGK-Cavβ interaction. Some RGK proteins also interact directly with the pore-forming α1 subunit of some types of VGCCs (Cavα1). Importantly, RGK proteins tonically inhibit VGCCs in native cells, regulating cardiac and neural functions. This minireview summarizes the mechanisms, molecular determinants, and physiological impact of RGK inhibition of VGCCs.

Rem uncouples excitation-contraction coupling in adult skeletal muscle fibers.

In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca(2+) channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca(2+) release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for β(1a) has been difficult to test because β(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, β(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca(2+) transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca(2+) store content. In contrast, a β(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca(2+) release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca(2+) release via its ability to interact with β(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β(1a) subunit in skeletal-type EC coupling.

A Low Affinity Cavb4a/Rem2 Interaction is Required for Inhibition of Cav2.1 Ca2+ Currents

RGK (Rem, Rad, Kir/Gem proteins) proteins, including Rem2, cause profound inhibition of high‐voltage activated Ca2+channels containing intracellular regulatory b subunits. All RGK proteins bind to Cavb subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies solution state nuclear magnetic resonance (NMR) and calorimetric techniques to map the binding site for Rem2 on human Cavb4a and to measure the binding affinity. Our experiments revealed two binding surfaces on the b4 GK domain contributing to a 156 μM Kd interaction: 1) a hydrophobic pocket surrounded by four residues, mutation of any of which completely disrupted binding; and 2) a nearby surface containing three residues that when individually mutated decreased affinity. Cav2.1 Ca2+currents were completely inhibited by Rem2 when co‐expressed with wild‐type Cavb4a, but were unaffected by Rem2 when co‐expressed with a Cavb4a site 1 mutant. These results provide direct evidence for a low affinity Rem2/Cavb4 interaction and its essential role in Cav2.1 inhibition by Rem2.

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel β4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory β subunits. All RGK proteins bind to voltage-gated Ca(2+) channel β subunit (Cavβ) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cavβ4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the β4 guanylate kinase domain contributing to a 156 ± 18 µM Kd interaction: a hydrophobic pocket lined by 4 critical residues (L173, N261, H262, and V303), mutation of any of which completely disrupted binding, and a nearby surface containing 3 residues (D206, L209, and D258) that when individually mutated decreased affinity. Voltage-gated Ca(2+) channel α1A subunit (Cav2.1) Ca(2+) currents were completely inhibited by Rem2 when co-expressed with wild-type Cavβ4a, but were unaffected by Rem2 when coexpressed with a Cavβ4a site 1 (L173A/V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cavβ4 interaction and show definitively that the interaction is required for Cav2.1 inhibition.

Ventricular L-Type Ca2+ Channels and Expression of RGK Proteins in Mouse Models Associated with Diabetes

Background: In a diabetic mouse model (db/db) we have shown reduced ICaL density with unchanged single-channel activity and reduced expression of the LTCC pore Cav1.2 (Pereira et al., Diabetes 2006;55:608-15). Of note, LTCC expression and function can be decreased by RGK proteins, including the diabetes-associated protein Rad. Aim of the study: In the present study, we investigate the association between cardiac Rad expression with the expression and function of ventricular LTCC in two mouse models with diabetes-related metabolic disturbances (leptin-deficient obese ob/ob mice and insulin receptor substrate 2 deficient IRS2-k.o. mice). Methods: We obtained expression of Rad and Cav1.2 protein (Western-blot) and mRNA (qRT-PCR) in murine ventricles and recorded whole-cell ICaL in freshly isolated ventricular myocytes. Results: The only significant change at the mRNA level was an increased Rad expression in IRS2-k.o. mice at 16 weeks of age (206±17%). In line with this finding ICaL density was significantly decreased (IRS2-k.o.: −7.8±0.8 pA/pF; wildtype: −10.9±0.9 pA/pF). At an age of 28 weeks, we found expression of both Rad and Cav1.2 protein to be significantly increased in ventricles from ob/ob mice compared to age-matched widtypes (273±23% and 159±14%, respectively) while ICaL density was unchanged (ob/ob: −8.4±0.4 pA/pF; wildtype: −8.9±0.5 pA/pF). Summary and discussion: Our data support the idea of Rad being involved in regulation of ICaL in diabetes. Regarding ICaL Rad seems to act either causal (ICaL decrease in IRS2-k.o. at 16 weeks) or compensatory (increased Cav1.2 expression but unchanged ICaL in ob/ob at 28 weeks). Differences observed between the two investigated diabetic mouse models might be explained by differences in the underlying pathomechanisms (lack of IRS2 vs. leptin deficiency, respectively).

RGK regulation of voltage-gated calcium channels.

Voltage-gated calcium channels (VGCCs) play critical roles in cardiac and skeletal muscle contractions, hormone and neurotransmitter release, as well as slower processes such as cell proliferation, differentiation, migration and death. Mutations in VGCCs lead to numerous cardiac, muscle and neurological disease, and their physiological function is tightly regulated by kinases, phosphatases, G-proteins, calmodulin and many other proteins. Fifteen years ago, RGK proteins were discovered as the most potent endogenous regulators of VGCCs. They are a family of monomeric GTPases (Rad, Rem, Rem2, and Gem/Kir), in the superfamily of Ras GTPases, and they have two known functions: regulation of cytoskeletal dynamics including dendritic arborization and inhibition of VGCCs. Here we review the mechanisms and molecular determinants of RGK-mediated VGCC inhibition, the physiological impact of this inhibition, and recent evidence linking the two known RGK functions.

Engineering Selectivity in RGK Protein Inhibition of Cav1/Cav2 Channels

High-voltage activated calcium channels (CaV1.1–CaV1.4; CaV2.1–CaV2.3) link electrical signals to vital physiological responses in excitable cells. Molecules that block CaV1/CaV2 channels are important therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are small Ras-like G-proteins that potently and indiscriminately inhibit all CaV1/CaV2 channels. The practical utility of RGKs as genetically-encoded CaV channel blockers would be vastly improved if it were possible to engineer versions that display selective inhibition of distinct CaV1/CaV2 isoforms. CaV1/CaV2 pore-forming α1 subunits require binding to auxiliary CaVβs to generate functional channels. All RGKs bind wild-type (wt) CaVβ and this interaction is disrupted in a mutated CaVβ (CaVβTM). We compared the ability of Rem and Gem to inhibit four distinct CaV1s (CaV1.2, CaV1.3, CaV2.1 and CaV2.2) reconstituted with either wtCaVβ or CaVβTM in HEK293 cells. While both Rem and Gem blocked all channels reconstituted with wtCaVβ, Rem uniquely suppressed CaV1.2+β2aTM channels, a signature of CaVβ-binding-independent inhibition. Using FRET analyses, chimeric Rem/Gem proteins, and electrophysiology we show that CaVβ-binding-independent inhibition of CaV1.2 involves direct interaction of Rem C-terminus with CaV1.2α1C N-terminus, and additionally requires the Rem nucleotide-binding domain. A mutant Rem that no longer interacts with CaVβ (Remβ-null) selectively inhibited CaV1.2+wtCaVβ channels. We further profiled the prevalence of β-binding-dependent and -independent mechanisms of inhibition by Rem2 and Rad across CaV1/CaV2 channel families. While Rem2 relied on CaVβ binding to inhibit all four CaVs tested, Rad displayed β-binding-independent inhibition of CaV1.2 and CaV2.2. Consistent with this, Remβ-null selectively inhibited CaV1.2 and CaV2.2 channels reconstituted with wtCaVβ. In summary, our results have revealed a latent capability of distinct RGK proteins to block particular CaV1/CaV2 channels in an α1-subunit-specific manner. We have exploited this feature to generate genetically-encoded CaV-isoform-selective inhibitors.

Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split.

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel β subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Cavβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.

RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (CaV1.1): (1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca2+ channel, and (3) voltage-sensor for slow depolarization-dependent Ca2+ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of CaV1.1. However, it is not known whether the latter function that has been attributed to CaV1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca2+ entry that is dependent on the voltage-sensing capability of CaV1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in “normal” mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFP-Rem, both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca2+ entry. Our observations provide the first evidence of modulation of this enigmatic Ca2+ entry pathway peculiar to skeletal muscle.

RGK Proteins Inhibit slow, Depolarization-Dependent CA2+ Entry into Cultured Myotubes

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (CaV1.1): 1) voltage-sensor for excitation-contraction (EC) coupling, 2) L-type Ca2+ channel, and 3) voltage-sensor for slow, depolarization-dependent Ca2+ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of CaV1.1. However, it is not known whether the latter function that has been attributed to CaV1.1 is subject to modulation by RGK proteins. The purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly activating, persistent Ca2+ entry that is dependent on the voltage-sensing capability of CaV1.1. To investigate this possibility, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in otherwise normal mouse myotubes and the abilities of each of these V-RGK proteins to inhibit EC coupling, L-type Ca2+ current and depolarization-dependent Ca2+ entry in myotubes were assessed using electrical field stimulation, whole-cell voltage-clamp and Ca2+ imaging, respectively. As shown previously for YFP-Rem, both EC coupling and L-type current density were substantially attenuated in developing myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca2+ entry (89%, 99% and 91% for V-Rad, V-Rem and V-Gem, respectively, relative to control). Thus, we provide the first evidence of modulation of this enigmatic type of Ca2+ entry peculiar to skeletal muscle. Moreover, the similar inhibitory effects of RGK proteins on both L-type current amplitude and slow Ca2+ entry provide further evidence that both modes of Ca2+ flux utilize a common pathway. Supported by AG038778 (to RAB).

Profiling Mechanisms of RGK Inhibition Across the Family of High-Voltage-Activated Cav1/Cav2 Calcium Channels

Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like monomeric G-proteins that powerfully potently inhibit all high-voltage-activated CaV1 and CaV2 calcium channels. Since all RGKs bind auxiliary CaVβs it was generally assumed that the RGK-β interaction is essential for CaV channel inhibition. Recently, using a mutated β (βTM) which), which selectively loses the ability to interact with RGKs, we reported that Rem inhibits CaV1.2 channels using both β-binding-dependent and direct α1-binding-dependent mechanisms (Yang et al, 2013, PLoS One, 7:e37079). Our aims here were twofold: (1) to identify determinants and mechanisms underlying direct Rem binding to, and inhibition of, CaV1.2 pore-forming α1C subunit;; (2) to profile the relative prevalence of β-binding-dependent and -independent mechanisms of inhibition across the RGK and CaV1/CaV2 channel families. Using a combination of FRET, co-immunoprecipitation assays, systematic truncations, and whole-cell electrophysiology we found that Rem C-terminus interacts with α1C N-terminus to inhibit CaV1.2 current (ICa,L) and gating charge. For profiling, we compared the impact of the four RGKs on currents through recombinant channels (CaV1.3, CaV2.1, CaV2.2) reconstituted with either wt β2a or β2a,TM, respectively. When reconstituted with wt β2a, all three channel types were strongly inhibited by each RGK. By contrast, when reconstituted with β2a,TM, CaV1.3 and CaV2.1 were completely refractory to all four RGKs indicating these channels display only CaVβ-binding-dependent mechanisms of inhibition. CaV2.2 channels reconstituted with β2a,TM displayed a strong inhibition solely to Rad, identifying a second example of CaVβ-binding-independent regulation of a CaV channel by an RGK protein. The results reveal latent capabilities of distinct RGKs to selectively inhibit particular CaV1/CaV2 channels in an isoform-specific manner. These dormant capabilities may be exploitable to develop novel genetically-encoded isoform-selective CaV1/CaV2 channel inhibitors.

The Amino-Termini of RGK Proteins Dictate the mode of L-Type Ca2+ Channel Inhibition in Adult Skeletal Muscle

Work in heterologous systems has revealed that RGK (Rad, Rem, Rem2, Gem/Kir) proteins inhibit L-type Ca2+ channels via three mechanisms: 1) reduction of membrane expression, 2) immobilization of the voltage-sensors, and 3) reduction of Po without impaired voltage-sensor movement. However, the question of which of these modes is (are) critical for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in mouse flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L- channel (CaV1.1). We found that Rad and Rem both potently inhibit excitation-contraction coupling and L-type current in FDB fibers. Charge movement was reduced (∼35%) in fibers overexpressing Rad, but charge movement in Rem-expressing fibers was identical to charge movement observed in control fibers. This unexpected result indicated that Rem supports inhibition solely through a mechanism which allows for translocation of CaV1.1's voltage-sensors whereas Rad utilizes at least one mode that limits voltage-sensor movement. Since Rad and Rem differ significantly only in their amino-termini, we constructed chimeras to probe the basis for the distinct specificities of Rad- and Rem-mediated inhibition. A chimera composed of the Rem amino-terminus and the Rad core/carboxyl-terminus inhibited L-current without reducing charge movement. Conversely, a chimera having the Rad amino-terminus fused to Rem core/carboxyl-terminus inhibited L-current with a concurrent reduction in charge movement. Thus, our work demonstrates that Rad and Rem exert their inhibitory effects on L-type channels in differentiated muscle fibers via distinct mechanisms. We have identified the amino-termini of Rad and Rem as the structural elements which dictate these specific modes of inhibition of CaV1.1. Supported by AG038778 (RAB) and 2T32AG000279-11 (Schwartz).