Shawn M. Crump

Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases

Rem, Rem2, Rad, and Gem/Kir (RGK) represent a distinct GTPase family with largely unknown physiological functions. We report here that both Rem and Rad bind directly to Ca2+ channel β-subunits (CaVβ) in vivo. No calcium currents are recorded from human embryonic kidney 293 cells coexpressing the L type Ca2+ channel subunits CaV1.2, CaVβ2a, and Rem or Rad, but CaV1.2 and CaVβ2a transfected cells elicit Ca2+ channel currents in the absence of these small G proteins. Importantly, CaV3 (T type) Ca2+ channels, which do not require accessory subunits for ionic current expression, are not inhibited by expression of Rem. Rem is expressed in primary skeletal myoblasts and, when overexpressed in C2C12 myoblasts, wild-type Rem inhibits L type Ca2+ channel activity. Deletion analysis demonstrates a critical role for the Rem C terminus in both regulation of functional Ca2+ channel expression and β-subunit association. These results suggest that all members of the RGK GTPase family, via direct interaction with auxiliary β-subunits, serve as regulators of L type Ca2+ channel activity. Thus, the RGK GTPase family may provide a mechanism for achieving cross talk between Ras-related GTPases and electrical signaling pathways.

Regulation of L-type Ca2+ Channel Activity and Insulin Secretion by the Rem2 GTPase

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions and are controlled by a diversity of intracellular signals. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. In this study, microarray analysis of the mouse insulinoma MIN6 cell line revealed that the transcription of Rem2 gene is strongly induced by exposure to high glucose, which was confirmed by real-time reverse transcriptase-PCR and RNase protection analysis. Because elevation of intracellular Ca2+ in pancreatic β-cells is essential for insulin secretion, we tested the hypothesis that Rem2 attenuates Ca2+ currents to regulate insulin secretion. Co-expression of Rem2 with CaV 1.2 or CaV1.3 L-type Ca + channels in a heterologous expression system completely inhibits de novo Ca2+ current expression. In addition, ectopic overexpression of Rem2 both inhibited L-type Ca2+ channel activity and prevented glucose-stimulated insulin secretion in pancreatic β-cell lines. Co-immunoprecipitation studies demonstrate that Rem2 associates with a variety of CaVβ subunits. Importantly, surface biotinylation studies demonstrate that the membrane distribution of Ca2+ channels was not reduced at a time when channel activity was potently inhibited by Rem2 expression, indicating that Rem2 modulates channel function without interfering with membrane trafficking. Taken together, these data suggest that inhibition of L-type Ca2+ channels by Rem2 signaling may represent a new and potentially important mechanism for regulating Ca2+-triggered exocytosis in hormone-secreting cells, including insulin secretion in pancreatic β-cells.

Analysis of the Complex between Ca2+ Channel β-Subunit and the Rem GTPase

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary β-subunit (CaVβ) binds a conserved domain (the α-interaction domain (AID)) of the pore-forming CaVα1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaVβ2a. The Rem interaction module is located in a ∼130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaVβ mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaVβ2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaVβ2a interaction is lower than that of AID for CaVβ2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaVβ2a with AID. Instead, CaVβ can simultaneously associate with both Rem and CaVα1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaVβ subunits with CaVα1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem·CaVβ·AID regulatory complex without the need to disrupt CaVα1·CaVβ association or alter CaVα1 expression at the plasma membrane.

L-type Calcium Channel Auto-Regulation of Transcription

L-type calcium channels (LTCC) impact the function of nearly all excitable cells. The classical LTCC function is to mediate trans-sarcolemmal Ca(2+) flux. This review focuses on the contribution of a mobile segment of the LTCC that regulates ion channel function, and also serves as a regulator of transcription in the nucleus. Specifically we highlight recent work demonstrating an auto-feedback regulatory pathway whereby the LTCC transcription factor regulates the LTCC. Also discussed is acute and long-term regulation of function by the LTCC-transcription regulator.

Rem-GTPase regulates cardiac myocyte L-type calcium current

Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca2+-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK–LTCC interactions is untested. Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca2+ current (ICa,L) via LTCC in murine cardiomyocytes. Methods and Results: Rem knockout mice (Rem−/−) were engineered, and ICa,L and Ca2+-handling properties were assessed. Rem−/− ventricular cardiomyocytes displayed increased ICa,L density. ICa,L activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type ICa,L. Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem−/−. Cell shortening was not significantly different. Increased ICa,L density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger ICa,L density, Rem−/− cardiomyocyte Ca2+ twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem−/− LTCC can account for the paradoxical decrease of Ca2+ transients. Conclusions: This is the first demonstration that loss of an RGK protein influences ICa,L in vivo in cardiac myocytes.

Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation.

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca2+ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca2+ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca2+ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of CaV1.2 (PCT), including the CB/IQ domain known to contribute to Ca2+/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the CaV1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca2+/CaM, and this effect is not due to Ca2+/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca2+ channel inhibition and slows the kinetics of Ca2+-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca2+ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca2+ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca2+ currents via modulation of Ca2+/CaM-mediated channel inactivation kinetics.

The cardiac L-type calcium channel distal carboxy terminus autoinhibition is regulated by calcium

The L-type calcium channel (LTCC) provides trigger Ca(2+) for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (I(Ba,L)) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca(2+) current (I(Ca,L)) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte I(Ca,L). We measured LTCC current and Ca(2+) transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and Ca(V)1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, Ca(V)1.2Δ1801, and a shorter deletion corresponding to well-studied construct, Ca(V)1.2Δ1733. DCT inhibited I(Ba,L) in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing Ca(V)1.2Δ1801 and Ca(V)1.2Δ1733. Ca(2+)-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of I(Ba,L) combined with Ca(2+)-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca(2+). We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca(2+), and show that DCT reduced diastolic Ca(2+) at low stimulation frequencies but spared high frequency Ca(2+) entry. DCT reduction of diastolic Ca(2+) and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca(2+) suggests that a physiological function of DCT is to increase the dynamic range of Ca(2+) transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

Analyses of Rem/RGK Signaling and Biological Activity

Rem (Rad and Gem related) is a member of the RGK family of Ras-related GTPases that also includes Rad, Rem2, and Gem/Kir. All RGK proteins share structural features that are distinct from other Ras-related proteins, including several nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C-terminal extension that contains regulatory sites that seem to control both subcellular location and function. Rem is known to modulate two distinct signal transduction pathways, regulating both cytoskeletal reorganization and voltage-gated Ca2+ channel activity. In this chapter, we summarize the experimental approaches used to characterize the interaction of Rem with 14-3-3 proteins and Ca2+ channel beta-subunits and describe electrophysiological analyses for characterizing Rem-mediated regulation of L-type Ca2+ channel activity.

The Cardiac L-Type Calcium Channel Distal Carboxyl Terminus is a Reverse use Dependent Inhibitor of Ca Current in Cardiomyocytes

Background. The LTCC distal C-terminus (DCT) is proteolytically cleaved, and re-associates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (IBa,L) in reconstituted channel complexes and mediates the fight or flight response in DCT knockout mice; yet the contribution of DCT to ICa,L in native systems is unexplored. This study examines the beat-to beat contribution of DCT to LTCC calcium homeostasis in native cardiomyocytes.Methods. We measured LTCC current with DCT co-expressed in cardiomyocytes. We also heterologously co-expressed DCT and CaV1.2 constructs with a stop corresponding to the predicted proteolytic cleavage site, Cav1.2Δ1801, and a shorter deletion corresponding to well-studied construct, Cav1.2Δ1733 along with CaVβ2 subunits.Results. DCT was found to inhibit IBa,L, but not ICa,L in cardiomyocytes. DCT blocked IBa,L carried by both Cav1.2Δ1801 and Cav1.2Δ1733 in HEK cells. However, DCT inhibited ICa,L carried by Cav1.2Δ1801 but not Cav1.2Δ1733 channels. As CaM and DCT have adjoining interaction sites on CaV1.2, we tested if exogenous CaM (CaMex) interferes with DCT current inhibition in cardiomyocytes and HEK systems. CaMex relieved DCT block in both systems and both channel variants. Addition of CaM1234 did not relieve DCT current inhibition or have an additive effect. DCT did not alter voltage dependence, or activation kinetics. The selective block of IBa,L suggested that DCT activity may be relieved under conditions of elevated Ca.Conclusions. DCT blockade of LTCC is apparent only when Ca is not the charge carrier. Therefore, our data motivates the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

The L-Type Calcium Channel C-Terminus is a Mobile Domain that Competes with Calmodulin Modulation of Calcium Current

The L-type Ca channel (CaV1.2) distal carboxyl-terminus (CCt) has multiple functions. CCt inhibits L-type calcium current (ICa,L), and is a mobile element that translocates to the nucleus where it regulates CaV1.2 transcription. CCt interacts with CaV1.2 in a similar domain as calmodulin (CaM). The purpose of this study is test the hypothesis that CaM and CCt compete for functional interaction with CaV1.2. ICa,L and barium current (IBa,L) was recorded from HEK 293 cells transfected with CaV1.2 + CaVbeta2a. This background was compared to cells additionally transfected with CaM and/or CCt. The CaV1.2 expressed was deleted at position 1733 (numbering based on rabbit sequence), and CCt corresponded to amino acids 1821-2171. ICa,L and IBa,L was recorded in each cell, and we compared the increase of current, the shift of activation midpoint, and current kinetics of ICa,L versus IBa,L within a given cell. Maximal conductance ratio Ca/Ba is ∼0.4 for CaV1.2+CaVbeta2a expression. Addition of CaM co-expression does not alter Ca /Ba conductance. CCt co-expression significantly increases the relative Ca /Ba ratio 2-fold, and this effect is reversed by CCt+CaM co-expression. Examination of the peak I(V) curves suggests that midpoint of activation was not affected, and ICa,L density is not different in for all transfection conditions. We conclude that CCt attenuation of conductance occurs only with Ba, and is consistent with a Ca alleviation of CCt block. Thus, CaM and Ca functionally compete to limit CCt auto-inhibition of CaV1.2 current.