CaVbeta Subunits Research Articles

Rem Inhibits Cav1.2 Channels using both Beta-Binding Dependent and Independent Mechanisms

Rad/Rem/Gem/Kir GTPases (RGKs) are the most potent known intracellular inhibitors of CaV1/CaV2 channels. Understanding their mechanism of action is critical not only for insights into their biological function but also may provide a blueprint for developing novel, useful protein CaV channel inhibitors. RGKs associate with auxiliary CaV beta subunits, but whether this interaction contributes to inhibition is elusive. We investigated the role of the RGK-beta interaction in Rem inhibition of reconstituted CaV1.2 channels, using a beta mutant (beta2aTM) selectively lacking binding to RGKs. Rem eliminated currents in wt beta2a-reconstituted channels (95% inhibition), but was significantly less potent (70% inhibition) in inhibiting beta2aTM-containing channels. Rem inhibits CaV1.2 channels using multiple mechanisms-decreasing channel number (N), open probability (Po), and gating charge (Qmax). The decreased N and Po (as demonstrated using a C1-domain-tagged Rem derivative that inducibly inhibits ICa,L when dynamically targeted to the membrane with PdBu, Figure) were eliminated in beta2aTM-reconstituted channels, indicating their beta-binding dependence. By contrast, reduced Qmax was retained in beta2aTM-reconstituted channels. The results shed new light on mechanisms of Rem inhibition of CaV1.2 channels and offer insights into rational development of novel bio-inspired CaV channel blockers.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ahnak1 modulates L-type Ca2+ channel inactivation of rodent cardiomyocytes

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.

G010 Regulation of voltage-dependent calcium channels by Nedd4-1

Calcium entry into excitable cells can be regulated by controlling both the activity of calcium channels and the amount of channels available at the plasma membrane. Little is known about their internalisation and degradation. One post-translational modification shown to be involved in membrane protein internalisation and subsequent degradation is the attachment of ubiquitin moieties by ubiquitin-ligases. Previously, it has been shown that cardiac ion channels such as Nav1.5 and KCNQ1 are down-regulated by ubiquitin-ligases of the Nedd4 family. These regulations involve the interaction between the PY motif of the target channels and the WW motif of the ubiquitin-ligases. Despite the absence of such PY motif in the cardiac voltage-gated calcium channel Cav1.2, we investigated whether this channel could by regulated in the same manner than other cardiac channels as previously described. We co-expressed, in HEK293 cells, L-type calcium channels and its two regulatory subunits Cavbeta and Cavalpha2delta1 together with ubiquitin-ligases, and examined by voltage-clamp whole-cell recordings the calcium current. We next determined by western blot and surface biotinylation assays the availability of the different subunits of calcium channels in total HEK293 lysates, and at the cell surface. Levels of ubiquitylation of the different subunits were assessed by pull-down GST-S5A and immunoprecipitation of Cav1.2 and its subunits. We found that co-expressing the ubiquitin-ligase Nedd4-1 significantly reduced Cav currents, and decreased Cavalpha and its subunit protein levels. This effect was Nedd4-1 specific since none of the other members of the Nedd4 family we tested produced a similar effect. We also found that the effect of Nedd4-1 was dependent on the co-expression of the Cavbeta subunit. No Nedd4-1-dependent increase in ubiquitylation of the Cavalpha protein was found; and unexpectedly, the two regulating subunits Cavbeta and Cavalpha2delta1 were detected to be deubiquitylated upon Nedd4-1 co-expression. Our data suggest that Nedd4-1 regulates the expression of Cav channels and its subunits by Nedd4-1 via an indirect mechanism constituting a new regulatory pathway to be determined. Further experiments will focus on the role of adrenergic receptors known to be ubiquitylated by Nedd4 and to bind to Cav1.2.

Determinants of the voltage dependence of G protein modulation within calcium channel beta subunits.

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gβγ-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVβ subunit with the CaVα1 subunit, when such interaction was prevented by a mutation in the α1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984–6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVβ subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVβ subunit that is required for this process. We have coexpressed the CaVβ subunit constructs with CaV2.2 and α2δ-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVβ subunit GK domains, from either β1b or β2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from β2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gβγ binding affinity, leading to greater tonic modulation by basal levels of Gβγ. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVβ subunit GK domains alone.

Novel CaV2.1 clone replicates many properties of Purkinje cell CaV2.1 current

The P-type calcium current is mediated by a voltage-sensing CaV2.1 alpha subunit in combination with modulatory auxiliary subunits. In Purkinje neurones, this current has distinctively slow inactivation kinetics that may depend on alternative splicing of the alpha subunit and/or association with different CaVbeta subunits. To better understand the molecular components of P-type calcium current, we cloned a CaV2.1 cDNA from total mouse brain. The full-length CaV2.1 isoform that we isolated (GenBank AY714490) contains sequences recently shown to be present in Purkinje neurones. In agreement with previously published work, the alternatively spliced amino acid V421, implicated in slow inactivation, was not encoded in AY714490 and was absent from reverse transcription-polymerase chain reaction products generated from single Purkinje cells. Next, we studied the expression of the four known mouse auxiliary CaVbeta2 isoforms in Purkinje neurones. Confirmation of the presence of CaVbeta2a in Purkinje cells, previously shown by others to slow CaV2.1 kinetics, led us to characterize its influence on current dynamics. We studied currents generated by the clone AY714490 coexpressed in tsA201 cells with four different CaVbeta subunits. In addition to the well-documented slowing of open-state inactivation kinetics, coexpression with the CaVbeta2a subunit also protected CaV2.1 channels from closed-state inactivation and prevented the channel from inactivating during physiological trains of action potential-like stimuli. This strong resistance to inactivation parallels the property of Purkinje neurone P-type currents and is suggestive of a role for CaVbeta2a in modulating the inactivation properties of P-type calcium currents in Purkinje neurones.

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 beta-subunit (CaV beta) binds a conserved domain (the alpha-interaction domain (AID)) of the pore-forming CaV alpha1 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 CaV beta2a. The Rem interaction module is located in a approximately 130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaV beta 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 CaV beta2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaV beta2a interaction is lower than that of AID for CaV beta2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaV beta2a with AID. Instead, CaV beta can simultaneously associate with both Rem and CaV alpha1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaV beta subunits with CaV alpha1 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 x CaV beta x AID regulatory complex without the need to disrupt CaV alpha1 x CaV beta association or alter CaV alpha1 expression at the plasma membrane.

Two PEST‐like motifs regulate Ca2+/calpain‐mediated cleavage of the CaVβ3 subunit and provide important determinants for neuronal Ca2+ channel activity

An increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVbeta subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVbeta subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVbeta3 contains two PEST motifs and undergoes Ca2+ -dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVbeta3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVbeta3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVbeta3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVbeta3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVbeta3 proteolysis may be an essential process for Ca2+ channel functional regulation.

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 beta-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 beta-cell lines. Co-immunoprecipitation studies demonstrate that Rem2 associates with a variety of CaVbeta 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 beta-cells.

Multiple determinants in voltage-dependent P/Q calcium channels control their retention in the endoplasmic reticulum.

Surface expression level of voltage-dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage-activated calcium channels requires the association of the pore-forming subunit, Cavalpha, with the auxiliary subunit, Cavbeta. In the absence of this auxiliary subunit, Cavalpha is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8 alpha chimeras, the molecular determinants of Cavalpha2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as 'ER retention'). This study demonstrates that the I-II loop of Cavalpha2.1 contains multiple ER-retention determinants beside the beta subunit association domain. In addition, the I-II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I-II loop, the N- and C-termini of Cavalpha2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low-threshold calcium channels, the I-II loop of Cavalpha1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i). a correct folding of Cavalpha in order to mask several internal ER-retention determinants and (ii). the association of other proteins, including the Cavbeta subunit, to suppress the remaining ER-retention determinants.