Analysis of the complex between Ca2+ channel beta-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.     

Negatively charged residues in the N-terminal of the AID helix confer slow voltage dependent inactivation gating to CaV1.2

The E462R mutation in the fifth position of the AID (alpha1 subunit interaction domain) region in the I-II linker is known to significantly accelerate voltage-dependent inactivation (VDI) kinetics of the L-type CaV1.2 channel, suggesting that the AID region could participate in a hinged-lid type inactivation mechanism in these channels. The recently solved crystal structures of the AID-CaVbeta regions in L-type CaV1.1 and CaV1.2 channels have shown that in addition to E462, positions occupied by Q458, Q459, E461, K465, L468, D469, and T472 in the rabbit CaV1.2 channel could also potentially contribute to a hinged-lid type mechanism. A mutational analysis of these residues shows that Q458A, Q459A, K465N, L468R, D469A, and T472D did not significantly alter VDI gating. In contrast, mutations of the negatively charged E461, E462, and D463 to neutral or positively charged residues increased VDI gating, suggesting that the cluster of negatively charged residues in the N-terminal end of the AID helix could account for the slower VDI kinetics of CaV1.2. A mutational analysis at position 462 (R, K, A, G, D, N, Q) further confirmed that E462R yielded faster VDI kinetics at +10 mV than any other residue with E462R > E462K approximately E462A > E462N > wild-type approximately E462Q approximately E462G > E462D (from the fastest to the slowest). E462R was also found to increase the VDI gating of the slow CEEE chimera that includes the I-II linker from CaV1.2 into a CaV2.3 background. The fast VDI kinetics of the CaV1.2 E462R and the CEEE + E462R mutants were abolished by the CaVbeta2a subunit and reinstated when using the nonpalmitoylated form of CaVbeta2a C3S + C4S (CaVbeta2a CS), confirming that CaVbeta2a and E462R modulate VDI through a common pathway, albeit in opposite directions. Altogether, these results highlight the unique role of E461, E462, and D463 in the I-II linker in the VDI gating of high-voltage activated CaV1.2 channels.     

Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain

Voltage-dependent calcium channels (VDCC) are multiprotein assemblies that regulate the entry of extracellular calcium into electrically excitable cells and serve as signal transduction centers. The alpha1 subunit forms the membrane pore while the intracellular beta subunit is responsible for trafficking of the channel to the plasma membrane and modulation of its electrophysiological properties. Crystallographic analyses of a beta subunit functional core alone and in complex with a alpha1 interaction domain (AID) peptide, the primary binding site of beta to the alpha1 subunit, reveal that beta represents a novel member of the MAGUK protein family. The findings illustrate how the guanylate kinase fold has been fashioned into a protein-protein interaction module by alteration of one of its substrate sites. Combined results indicate that the AID peptide undergoes a helical transition in binding to beta. We outline the mechanistic implications for understanding the beta subunit's broad regulatory role of the VDCC, particularly via the AID.     

Molecular determinants of inactivation within the I-II linker of alpha1E (CaV2.3) calcium channels

Voltage-dependent inactivation of CaV2.3 channels was investigated using point mutations in the beta-subunit-binding site (AID) of the I-II linker. The quintuple mutant alpha1E N381K + R384L + A385D + D388T + K389Q (NRADK-KLDTQ) inactivated like the wild-type alpha1E. In contrast, mutations of alpha1E at position R378 (position 5 of AID) into negatively charged residues Glu (E) or Asp (D) significantly slowed inactivation kinetics and shifted the voltage dependence of inactivation to more positive voltages. When co-injected with beta3, R378E inactivated with tau(inact) = 538 +/- 54 ms (n = 14) as compared with 74 +/- 4 ms (n = 21) for alpha1E (p < 0.001) with a mid-potential of inactivation E(0.5) = -44 +/- 2 mV (n = 10) for R378E as compared with E(0.5) = -64 +/- 3 mV (n = 9) for alpha1E. A series of mutations at position R378 suggest that positively charged residues could promote voltage-dependent inactivation. R378K behaved like the wild-type alpha1E whereas R378Q displayed intermediate inactivation kinetics. The reverse mutation E462R in the L-type alpha1C (CaV1.2) produced channels with inactivation properties comparable to alpha1E R378E. Hence, position 5 of the AID motif in the I-II linker could play a significant role in the inactivation of Ca(V)1.2 and CaV2.3 channels.     

Functional expression and characterization of a voltage-gated CaV1.3 (alpha1D) calcium channel subunit from an insulin-secreting cell line.

L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type alpha1-subunits in pancreatic beta-cells is of the neuroendocrine subtype (CaV1.3 or alpha1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or alpha1C-a) after expression together with alpha2delta- and beta3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound G(i)/G(o)-coupled GPCRs whereas a neuronal CaV2.2 (or alpha1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by G(i)/G(o)-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.     

The alpha1-beta-subunit interaction that modulates calcium channel activity is reversible and requires a competent alpha-interaction domain

High voltage-gated calcium channels consist of a pore-forming subunit (alpha(1)) and three nonhomologous subunits (alpha(2)/delta, beta, and gamma). Although it is well established that the beta-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the alpha(1)-subunit remains controversial. Here, we address this issue by examining the effect of purified beta(2a) protein on Ca(V)1.2 and Ca(V)2.3 channels expressed in Xenopus oocytes. The beta(2a)-subunit binds to the alpha(1)-interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of Ca(V)1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the alpha(1)-beta interaction through the AID site can take place at the plasma membrane. Furthermore, injection of beta(2a) protein inhibited inactivation of Ca(V)2.3 channels and converted fast inactivating Ca(V)2.3/beta(1b) channels to slow inactivating channels. Inhibition of inactivation required larger concentration of beta(2a) in oocytes expressing Ca(V)2.3/beta(1b) channels than expressing Ca(V)2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the alpha(1)-beta interaction is reversible in intact cells and defines calcium channels beta-subunits as regulatory proteins rather than stoichiometric subunits.     

Direct inhibition of the interaction between alpha-interaction domain and beta-interaction domain of voltage-dependent Ca2+ channels by Gem

The Ras-related small G-protein Gem regulates voltage-dependent Ca2+ channels (VDCCs) through interaction with the beta-subunit of the VDCC. This action of Gem is mediated by regulated alpha1-subunit expression at the plasma membrane. In the present study, we examined the mechanism of the inhibition of VDCC activity by Gem. The beta-interaction domain (BID) of the beta-subunit, which specifically interacts with the alpha-interaction domain (AID) of the alpha1-subunit, is shown to be essential for the interaction between Gem and beta-subunits. In addition, the AID peptide inhibited interaction between Gem and beta-subunits in a dose-dependent manner. GemS88N mutant, which has low binding affinity for guanine nucleotide, did not interact with beta-subunits, allowing alpha1-subunit expression at the plasma membrane. This inhibitory effect of wild-type Gem on VDCC activity was reduced in cells expressing GemS88N. The overexpression of wild-type Gem in pancreatic beta-cell line MIN6 cells suppressed Ca2+-triggered secretion, whereas overexpression of GemS88N induced Ca2+-triggered secretion to control level. These results suggest that GTPase activity of Gem is required for the binding of Gem to BID that regulates VDCC activity through interaction with AID.     

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.     

Analysis of the Rem2 - voltage dependant calcium channel beta subunit interaction and Rem2 interaction with phosphorylated phosphatidylinositide lipids

Voltage dependant calcium channels (VDCC) play a critical role in coupling electrical excitability to important physiological events such as secretion by neuronal and endocrine cells. Rem2, a GTPase restricted to neuroendocrine cell types, regulates VDCC activity by a mechanism that involves interaction with the VDCC beta subunit (Ca(V)beta). Mapping studies reveal that Rem2 binds to the guanylate kinase domain (GK) of the Ca(V)beta subunit that also contains the high affinity binding site for the pore forming and voltage sensing VDCC alpha subunit (Ca(V)alpha) interaction domain (AID). Moreover, fine mapping indicates that Rem2 binds to the GK domain in a region distinct from the AID interaction site, and competitive inhibition studies reveal that Rem2 does not disrupt Ca(V)alpha - Ca(V)beta binding. Instead, the Ca(V)beta subunit appears to serve a scaffolding function, simultaneously binding both Rem2 and AID. Previous studies have found that in addition to Ca(V)beta binding, Rem2 must be localized to the plasma membrane to inhibit VDCC function. Plasma membrane localization requires the C-terminus of Rem2 and binding studies indicate that this domain directs phosphorylated phosphatidylinositide (PIP) lipids association. Plasma membrane localization may provide a unique point of regulation since the ability of Rem2 to bind PIP lipids is inhibited by the phosphoserine dependant binding of 14-3-3 proteins. Thus, in addition to Ca(V)beta binding, VDCC blockade by Rem2 is likely to be controlled by both the localized concentration of membrane PIP lipids and direct 14-3-3 binding to the Rem2 C-terminus.     

Reversibility of the Ca(2+) channel alpha(1)-beta subunit interaction

The auxiliary beta subunit importantly regulates voltage-dependent Ca(2+) channel activity through an interaction with the AID domain, a binding site located in the cytoplasmic I-II linker of the ion-conducting alpha(1) subunit. In the present study, we used two synthetic peptides corresponding to partial sequences of the I-II linker of alpha(1A) (AID(A)-peptides) as tools to disrupt the alpha(1)-beta interaction. In vitro binding experiments confirmed that these peptides exhibit a reasonable affinity to the neuronal beta(3) subunit to serve this purpose, although they failed to prevent immunoprecipitation of native N- and P/Q-type channels by anti-beta(3) antibodies. Together, our results (i) provide evidence for the reversibility of channel subunit association suggesting that the disruption of the alpha(1)-beta interaction may be a possible mechanism for Ca(2+) channel regulation in vivo, and (ii) support a model whereby the alpha(1)-beta association is based on multiple interaction sites.     

The voltage-dependent calcium channel beta subunit contains two stable interacting domains

Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.     

A new beta subtype-specific interaction in alpha1A subunit controls P/Q-type Ca2+ channel activation

The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.     

Zn2+ sensitivity of high- and low-voltage activated calcium channels

The essential cation zinc (Zn2+) blocks voltage-dependent calcium channels in several cell types, which exhibit different sensitivities to Zn2+. The specificity of the Zn2+ effect on voltage-dependent calcium channel subtypes has not been systematically investigated. In this study, we used a transient protein expression system to determine the Zn2+ effect on low- and high-voltage activated channels. We found that in Ba2+, the IC50 value of Zn2+ was alpha1-subunit-dependent with lowest value for CaV1.2, and highest for CaV3.1; the sensitivity of the channels to Zn2+ was approximately ranked as CaV1.2>CaV3.2>CaV2.3>CaV2.2=CaV 2.1>or=CaV3.3=CaV3.1. Although the CaV2.2 and CaV3.1 channels had similar IC50 for Zn2+ in Ba2+, the CaV2.2, but not CaV3.1 channels, had approximately 10-fold higher IC50 to Zn2+ in Ca2+. The reduced sensitivity of CaV2.2 channels to Zn2+ in Ca2+ was partially reversed by disrupting a putative EF-hand motif located external to the selectivity filter EEEE locus. Thus, our findings support the notion that the Zn2+ block, mediated by multiple mechanisms, may depend on conformational changes surrounding the alpha1 pore regions. These findings provide fundamental insights into the mechanism underlying the inhibitory effect of zinc on various Ca2+ channel subtypes.     

Identification of caveolin-1-interacting sites in neuronal nitric-oxide synthase. Molecular mechanism for inhibition of NO formation

Caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (CaV1p1; residues (82-101)) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an IC(50) value of 1.8 microM. Mutations of Phe(584) and Trp(587) within a caveolin binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences in the mechanism of inhibition by caveolin for nNOS as compared with those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. We also examined the effects of CaV1p1 on an autoinhibitory domain deletion mutant (Delta40) and a C-terminal truncation mutant (DeltaC33), both of which are able to form NO in the absence of CaM, unlike the wild-type enzyme. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaM-independent, NO formation activities of both Delta40 and DeltaC33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.     

The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel CaV1.2

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.     

Molecular determinants of modulation of CaV2.1 channels by visinin-like protein 2

CaV2.1 channels, which conduct P/Q-type Ca2+ currents, initiate synaptic transmission at most synapses in the central nervous system. Ca2+/calmodulin-dependent facilitation and inactivation of these channels contributes to short-term facilitation and depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin (CaM) from its binding site, differentially regulate CaV2.1 channels, and contribute to the diversity of short-term synaptic plasticity. The neuronal calcium sensor protein visinin-like protein 2 (VILIP-2) inhibits inactivation and enhances facilitation of CaV2.1 channels. Here we examine the molecular determinants for differential regulation of CaV2.1 channels by VILIP-2 and CaM by construction and functional analysis of chimeras in which the functional domains of VILIP-2 are substituted in CaM. Our results show that the N-terminal domain, including its myristoylation site, the central α-helix, and the C-terminal lobe containing EF-hands 3 and 4 of VILIP-2 are sufficient to transfer its regulatory properties to CaM. This regulation by VILIP-2 requires binding to the IQ-like domain of CaV2.1 channels. Our results identify the essential molecular determinants of differential regulation of CaV2.1 channels by VILIP-2 and define the molecular code that these proteins use to control short-term synaptic plasticity.