RGK GTPases Research Articles

Inactivation influences the extent of inhibition of voltage-gated Ca+2 channels by Gem-implications for channelopathies.

Voltage-gated Ca2+ channels (VGCC) directly control muscle contraction and neurotransmitter release, and slower processes such as cell differentiation, migration, and death. They are potently inhibited by RGK GTP-ases (Rem, Rem2, Rad, and Gem/Kir), which decrease Ca2+ channel membrane expression, as well as directly inhibit membrane-resident channels. The mechanisms of membrane-resident channel inhibition are difficult to study because RGK-overexpression causes complete or near complete channel inhibition. Using titrated levels of Gem expression in Xenopus oocytes to inhibit WT P/Q-type calcium channels by ∼50%, we show that inhibition is dependent on channel inactivation. Interestingly, fast-inactivating channels, including Familial Hemiplegic Migraine mutants, are more potently inhibited than WT channels, while slow-inactivating channels, such as those expressed with the Cavβ2a auxiliary subunit, are spared. We found similar results in L-type channels, and, remarkably, Timothy Syndrome mutant channels were insensitive to Gem inhibition. Further results suggest that RGKs slow channel recovery from inactivation and further implicate RGKs as likely modulating factors in channelopathies.

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.

Inactivation Regulates RGK-Mediated Inhibition of Voltage-Gated Calcium Channels

Voltage-gated Ca2+ channels (VGCC) directly control muscle contraction and neurotransmitter release, and slower processes such as cell differentiation, migration, and death. They are potently inhibited by RGK GTP-ases (Rem1, Rem2, Rad and Gem/Kir), which decrease Ca2+ channel membrane expression, as well as directly inhibit membrane-resident channels. The mechanisms of membrane-resident channel inhibition are difficult to study because RGK-overexpression causes complete or near complete channel inhibition. Using titrated levels of RGK expression in Xenopus oocytes that inhibit WT P/Q-type calcium channels by ∼50%, we show that inhibition depends on channel inactivation. Interestingly, fast-inactivating channels, including Familial Hemiplegic Migraine mutants, are more potently inhibited than WT channels, while slow-inactivating channels, such as those expressed with the β2a auxiliary subunit, are spared. We found similar results in L-type channels, and, remarkably, an insensitivity of Timothy Syndrome mutants to RGK inhibition. Further results suggest that RGKs slow channel recovery from inactivation and identify RGKs as potential modulating factors in channelopathies. Finally, our results confirm a previously proposed immobilization of calcium channel voltage sensors by Rad, which we were now able to observe in electrophysiological experiments with titrated levels of Rad. Thus, RGK-mediated inhibition is a much subtler affair than previously thought, and physiological conditions can control the levels of inhibition.

Optogenetic Control of Voltage-Gated Calcium Channels.

Voltage‐gated Ca2+ (CaV) channels mediate Ca2+ entry into excitable cells to regulate a myriad of cellular events following membrane depolarization. We report the engineering of RGK GTPases, a class of genetically encoded CaV channel modulators, to enable photo‐tunable modulation of CaV channel activity in excitable mammalian cells. This optogenetic tool (designated optoRGK) tailored for CaV channels could find broad applications in interrogating a wide range of CaV‐mediated physiological processes.

Optogenetic Control of Voltage‐Gated Calcium Channels

AbstractVoltage‐gated Ca2+ (CaV) channels mediate Ca2+ entry into excitable cells to regulate a myriad of cellular events following membrane depolarization. We report the engineering of RGK GTPases, a class of genetically encoded CaV channel modulators, to enable photo‐tunable modulation of CaV channel activity in excitable mammalian cells. This optogenetic tool (designated optoRGK) tailored for CaV channels could find broad applications in interrogating a wide range of CaV‐mediated physiological processes.

Regulation of neuronal high-voltage activated CaV2 Ca2+ channels by the small GTPase RhoA

High-Voltage-Activated (HVA) Ca2+ channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca2+ channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca2+ channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba2+ currents through CaV2.1, CaV2.2 and CaV2.3 Ca2+ channels independently of CaVβ subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca2+ current amplitude. Altogether our data revealed that HVA Ca2+ channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired.

Nerve injury induces a Gem-GTPase-dependent downregulation of P/Q-type Ca2+ channels contributing to neurite plasticity in dorsal root ganglion neurons.

Small RGK GTPases, Rad, Gem, Rem1, and Rem2, are potent inhibitors of high-voltage-activated (HVA) Ca(2+) channels expressed in heterologous expression systems. However, the role of this regulation has never been clearly demonstrated in the nervous system. Using transcriptional analysis, we show that peripheral nerve injury specifically upregulates Gem in mice dorsal root ganglia. Following nerve injury, protein expression was increased in ganglia and peripheral nerve, mostly under its phosphorylated form. This was confirmed in situ and in vitro in dorsal root ganglia sensory neurons. Knockdown of endogenous Gem, using specific small-interfering RNA (siRNA), increased the HVA Ca(2+) current only in the large-somatic-sized neurons. Combining pharmacological analysis of the HVA Ca(2+) currents together with Gem siRNA-transfection of larger sensory neurons, we demonstrate that only the P/Q-type Ca(2+) channels were enhanced. In vitro analysis of Gem affinity to various CaVβx-CaV2.x complexes and immunocytochemical studies of Gem and CaVβ expression in sensory neurons suggest that the specific inhibition of the P/Q channels relies on both the regionalized upregulation of Gem and the higher sensitivity of the endogenous CaV2.1-CaVβ4 pair in a subset of sensory neurons including the proprioceptors. Finally, pharmacological inhibition of P/Q-type Ca(2+) current reduces neurite branching of regenerating axotomized neurons. Taken together, the present results indicate that a Gem-dependent P/Q-type Ca(2+) current inhibition may contribute to general homeostatic mechanisms following a peripheral nerve injury.

Distinct RGK GTPases Differentially Use α1- and Auxiliary β-Binding-Dependent Mechanisms to Inhibit CaV1.2/CaV2.2 Channels

CaV1/CaV2 channels, comprised of pore-forming α1 and auxiliary (β,α2δ) subunits, control diverse biological responses in excitable cells. Molecules blocking CaV1/CaV2 channel currents (I Ca) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit CaV1/CaV2 channels. Understanding how RGKs block I Ca is critical for insights into their physiological function, and may provide design principles for developing novel CaV1/CaV2 channel inhibitors. The RGK binding sites within CaV1/CaV2 channel complexes responsible for I Ca inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind β subunits, but it is unknown if and how this interaction contributes to I Ca inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant CaV1.2 channels, using a mutated β (β2aTM) selectively lacking RGK binding. Rem blocked β2aTM-reconstituted channels (74% inhibition) less potently than channels containing wild-type β2a (96% inhibition), suggesting the prevalence of both β-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of CaV1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to β. We identified a novel Rem binding site in CaV1.2 α1C N-terminus that mediated β-binding-independent inhibition. The CaV2.2 α1B subunit lacks the Rem binding site in the N-terminus and displays a solely β-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs— while Rem and Rad use both β-binding-dependent and independent mechanisms, Gem and Rem2 use only a β-binding-dependent method to inhibit CaV1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of CaV1.2/CaV2.2 channels by distinct RGK GTPases.

The β Subunit of Voltage-Gated Ca2+Channels

Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entryways for Ca(2+) in excitable cells are high-voltage activated (HVA) Ca(2+) channels. These are plasma membrane proteins composed of several subunits, including α(1), α(2)δ, β, and γ. Although the principal α(1) subunit (Ca(v)α(1)) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Ca(v)β) plays an essential role in regulating the surface expression and gating properties of HVA Ca(2+) channels. Ca(v)β is also crucial for the modulation of HVA Ca(2+) channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca(2+) channels by binding to Ca(v)β. There are also indications that Ca(v)β may carry out Ca(2+) channel-independent functions, including directly regulating gene transcription. All four subtypes of Ca(v)β, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Ca(v)βs reveal how they interact with Ca(v)α(1), open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Ca(v)β, with both a historical perspective as well as an emphasis on recent advances.

Molecular Mechanisms, and Selective Pharmacological Rescue, of Rem-Inhibited Ca V 1.2 Channels in Heart

In heart, Ca(2+) entering myocytes via Ca(V)1.2 channels controls essential functions, including excitation-contraction coupling, action potential duration, and gene expression. RGK GTPases (Rad/Rem/Rem2/Gem/Kir sub-family of Ras-like GTPases) potently inhibit Ca(V)1.2 channels, an effect that may figure prominently in cardiac Ca(2+) homeostasis under physiological and disease conditions. To define the mechanisms and molecular determinants underlying Rem GTPase inhibition of Ca(V)1.2 channels in heart and to determine whether such inhibited channels can be pharmacologically rescued. Overexpressing Rem in adult guinea pig heart cells dramatically depresses L-type calcium current (I(Ca,L)) ( approximately 90% inhibition) and moderately reduces maximum gating charge (Q(max)) (33%), without appreciably diminishing the physical number of channels in the membrane. Rem-inhibited Ca(V)1.2 channels were supramodulated by BAY K 8644 (10-fold increase) compared to control channels (3-fold increase). However, Rem prevented protein kinase A-mediated upregulation of I(Ca,L), an effect achieved without disrupting the sympathetic signaling cascade because protein kinase A modulation of I(KS) (slow component of the delayed rectifier potassium current) remained intact. In accord with its functional impact on I(Ca,L), Rem selectively prevented protein kinase A- but not BAY K 8644-induced prolongation of the cardiac action potential duration. A GTP-binding-deficient Rem[T94N] mutant was functionally inert with respect to I(Ca,L) inhibition. A chimeric construct, Rem(265)-H, featuring a swap of the Rem C-terminal tail for the analogous domain from H-Ras, inhibited I(Ca,L) and Q(max) to the same extent as wild-type Rem, despite lacking the capacity to autonomously localize to the sarcolemma. Rem predominantly inhibits I(Ca,L) in heart by arresting surface Ca(V)1.2 channels in a low open probability gating mode, rather than by interfering with channel trafficking. Moreover, Rem-inhibited Ca(V)1.2 channels can be selectively rescued by BAY K 8644 but not protein kinase A-dependent phosphorylation. Contrary to findings in reconstituted systems, Rem-induced ablation of cardiac I(Ca,L) requires GTP-binding, but not membrane-targeting of the nucleotide binding domain. These findings provide a different perspective on the molecular mechanisms and structural determinants underlying RGK GTPase inhibition of Ca(V)1.2 channels in heart, and suggest new (patho)physiological dimensions of this crosstalk.

RGK GTPase‐dependent CaV2.1 Ca2+channel inhibition is independent of CaVβ–subunit‐induced current potentiation

RGK (Rad-Gem-Rem) GTPases have been described as potent negative regulators of the Ca(2+) influx via high-threshold voltage-activated Ca(2+) channels. Recent work, mostly performed on Ca(V)1.2 Ca(2+) channels, has highlighted the crucial role played by the channel auxiliary Ca(V)beta subunits and identified several GTPase and beta-subunit protein domains involved in this regulation. We now extend these conclusions by producing the first complete characterization of the effects of Gem, Rem, and Rem2 on the neuronal Ca(V)2.1 Ca(2+) channels expressed with Ca(V)beta(1) or Ca(V)beta(2) subunits. Current inhibition is limited to a decrease in amplitude with no modification in the voltage dependence or kinetics of the current. We demonstrate that this inhibition can occur for Ca(V)beta constructs with impaired capacity to induce current potentiation, but that it is lost for Ca(V)beta constructs deleted for their beta-interaction domain. The RGK C-terminal last approximately 80 amino acids are sufficient to allow potent current inhibition and in vivo beta-subunit/Gem interaction. Interestingly, although Gem and Gem carboxy-terminus induce a completely different pattern of beta-subunit cellular localization, they both potently inhibit Ca(V)2.1 channels. These data therefore set the status of neuronal Ca(V)2.1 Ca(2+) channel inhibition by RGK GTPases, emphasizing the role of short amino acid sequences of both proteins in beta-subunit binding and channel inhibition and revealing a new mechanism for channel inhibition.

Rem Selectively Abolishes β1-adrenergic Regulation Of CaV1.2 Channels In Heart

β-adrenergic modulation of cardiac CaV1.2 channels is critical for sympathetic regulation of the heartbeat, and its disruption is a harmful hallmark of heart failure. RGK (Rem, Rem2, Rad, Gem/Kir) GTPases potently inhibit CaV channels by interacting with their auxiliary β subunits. Intriguingly, RGK proteins are present in heart, and their levels are elevated in heart failure. We examined the impact of the RGK GTPase, Rem, on CaV1.2 channels in heart cells and assessed whether there was crosstalk with the β-adrenergic modulation of the channel. Cultured adult guinea pig ventricular myocytes expressed robust CaV1.2 currents (ICa,L) (15.08 pA/pF) and responded to β1-adrenergic activation (1 μM isoproterenol + 1 μM ICI118,551) with a sharp, three-fold increase in current density. Isochronal cardiac cells expressing YFP-Rem, achieved through adenovirus infection, displayed a markedly lower basal current density (5.85 pA/pF). Nevertheless, the effect of Rem in heart is quantitatively smaller than seen in recombinant channels expressed in HEK 293 cells, which feature a virtual ablation of ICa,L. Surprisingly, the remaining Rem-insensitive ICa,L in guinea pig heart cells was essentially unresponsive to β1-adrenergic stimulation. This was not due to disruption of the signaling pathway because isoproterenol-mediated increase in cardiac IKs remained unchanged. Intriguingly, the Rem insensitive ICa,L remained responsive to forskolin. These results reveal an unexpected crosstalk between RGK GTPases and β-adrenergic signaling pathway at the level of cardiac ICa,L, and suggests that Rem selectively inhibits spatially distinct CaV1.2 channels in single heart cells.

Multiple Mechanisms and Determinants Underlie Rem Inhibition of Voltage-dependent Calcium (CaV) Channels

RGK GTPases potently inhibit CaV channels, an effect with critical (patho)physiological implications and potential biotechnology applications. Mechanisms of how RGK proteins inhibit ICa are poorly understood. Critical ambiguities surround: (1) whether inhibition occurs exclusively by either reducing the number of channels (N) at the membrane, or diminishing the activity (Po) of channels in the membrane, and (2) the role of RGK protein sub-cellular localization. Whole-cell experiments on recombinant CaV1.2 channels indicated that Rem inhibited ICa by two distinct effects: a decrease in N, as gauged by gating charge measurements, and a reduction in effective Po of channels at the membrane. These two effects were kinetically distinguishable. Replacing the membrane-targeting C-terminus of Rem with the PKCγ C1 domain permitted acutely inducible membrane translocation of Rem[265]-C1PKCγ and subsequent ICa inhibition by PdBu. Pdbu activation of Rem[265]-C1PKCγ acutely (seconds) inhibited ICa by selectively reducing effective Po, while the decrease in N occurred after a longer time scale (hours). A prevailing paradigm is that membrane localization is essential for RGK GTPase inhibition of ICa. Nevertheless, a Rem C-terminus point mutant, Rem[L271G], that distributes to the nucleus and cytosol, significantly inhibits ICa leading to a suggestion that nuclear targeting represents an alternative mechanistic mode of action for Rem. However, Rem[L271G] exclusively targeted to the nucleus using a nucleus localization sequence had no impact on ICa. By contrast, an exclusively cytosolic Rem[L271G], achieved using a nucleus export sequence, essentially ablated ICa, demonstrating a clear-cut exception to the importance of membrane-targeting for RGK GTPase action on ICa. Our results reveal that the exceptional potency of Rem in inhibiting ICa is achieved via an unusual multiplicity of mechanisms and structural determinants.

Plasma Membrane Targeting Is Essential for Rem-mediated Ca2+ Channel Inhibition

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca(2+) channels and a known interacting partner for Ca(2+) channel accessory beta subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that Ca(V)beta association is required. Previous work has shown that a C-terminal truncation of Rem (Rem-(1-265)) displays reduced in vivo binding to membrane-localized beta 2a and lacks channel regulatory function. In this paper, we describe a role for the Rem C terminus in plasma membrane localization through association with phosphatidylinositol lipids. Moreover, Rem-(1-265) can associate with beta 2a in vitro and beta 1b in vivo, suggesting that the C terminus does not directly participate in Ca(V)beta association. Despite demonstrated beta 1b binding, Rem-(1-265) was not capable of regulating a Ca(V)1.2-beta 1b channel complex, indicating that beta subunit binding is not sufficient for channel regulation. However, fusion of the CAAX domain from K-Ras4B or H-Ras to the Rem-(1-265) C terminus restored membrane localization and Ca(2+) channel regulation, suggesting that beta binding and membrane localization are independent events required for channel inhibition.

Dose-dependent and Isoform-specific Modulation of Ca2+ Channels by RGK GTPases

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca2+ influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs—Gem, Rem, and Rem2—decreased CaV1.2 Ca2+ current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca2+ currents and affect Ca2+ influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca2+ channel accessory β subunit's interaction with its high affinity binding site in the pore-forming α1C subunit (AID) from its other putative contact sites by utilizing an α1C•β3 concatemer in which the AID was mutated to prevent β subunit interaction. This mutant concatemer generated currents with all the hallmarks of β subunit modulation, demonstrating that AID-β–independent interactions are sufficient for β subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca2+ channel current modulation.