Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K- ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K- pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K- pumps.     

Four-turn alpha-helical segment prevents surface expression of the auxiliary hbeta2 subunit of BK-type channel

Large conductance, voltage- and Ca2+-activated K+ (BK) channels encoded by the mslo alpha and beta2 subunits exist abundantly in rat chromaffin cells, pancreatic beta cells, and DRG neurons. The extracellular loop of hbeta2 acting as the channel regulator influences the rectification and toxin sensitivity of BK channels, and the inactivation domain at its N terminus induces rapid inactivation. However, the regulatory mechanism, especially the trafficking mechanism of hbeta2, is still unknown. With the help of immunofluorescence and patch clamp techniques, we determine that the hbeta2 subunit alone resides in the endoplasmic reticulum, suggesting that trafficking mechanism of hbeta2 differs from that of hbeta1 opposite to what we predicted previously. We further demonstrate that a four-turn alpha helical segment at the N terminus of hbeta2 prevents the surface expression of hbeta2, that is, the helical segment itself is a retention signal. Using the c-Myc epitope-tagged extracellular loop of hbeta2, we reveal that the most accessible site by antibody is located at the middle of the extracellular loop, which might provide clues to understand how the auxiliary beta subunits regulates the toxin sensitivity and the rectification of BK-type channels.     

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.     

A mutation in the beta interaction domain of the Ca(2+) channel alpha(1C) subunit reduces the affinity of the (+)-[(3)H]isradipine binding site

The molecular mechanisms of how alpha(1) and beta subunits of voltage-gated Ca(2+) channels interact with one another are still controversial. Here we show that despite a mutation in the beta interaction domain that has previously been shown to disrupt binding, alpha(1C)Y467S and beta(1a-myc) still formed immunoprecipitable complexes when coexpressed in tsA201 cells. However, the alpha(1C)Y467S-beta(1a-myc) complexes had a decreased affinity to (+)-[(3)H]isradipine. This indicates that the beta interaction domain in the I-II loop of the alpha(1) subunit is not merely an anchor required for the functional interaction of the two Ca(2+) channel subunits but is itself part of the effector pathway for beta-induced channel modulation.     

Endoplasmic reticulum quality control of oligomeric membrane proteins: topogenic determinants involved in the degradation of the unassembled Na,K-ATPase alpha subunit and in its stabilization by beta subunit assembly

The molecular nature of determinants that mediate degradation of unassembled, polytopic subunits of oligomeric membrane proteins and their stabilization after partner subunit assembly is largely unknown. Expressing truncated Na,K-ATPase alpha subunits alone or together with beta subunits, we find that in unassembled alpha subunits neither the four N-terminal transmembrane segments acting as efficient alternating signal anchor-stop transfer sequences nor the large, central cytoplasmic loop exposes any degradation signal, whereas poor membrane insertion efficiency of C-terminal membrane domains M5, M7, and M9 coincides with the transient exposure of degradation signals to the cytoplasmic side. beta assembly with an alpha domain comprising at least D902 up to Y910 in the extracytoplasmic M7/M8 loop is necessary to stabilize Na,K-ATPase alpha subunits by favoring M7/M8 membrane pair formation and by protecting a degradation signal recognized from the endoplasmic reticulum (ER) lumenal side. Thus our results suggest that ER degradation of Na,K-ATPase alpha subunits is 1) mainly mediated by folding defects caused by inefficient membrane insertion of certain membrane domains, 2) a multistep process, which involves proteolytic and/or chaperone components acting from the ER lumenal side in addition to cytosolic, proteasome-related factors, and 3) prevented by partner subunit assembly because of direct protection and retrieval of degradation signals from the cytoplasm to the ER lumenal side. These results likely represent a paradigm for the ER quality control of unassembled, polytopic subunits of oligomeric membrane proteins.     

Role of the C terminus of the alpha 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels

We have previously demonstrated that formation of a complex between L-type calcium (Ca(2+)) channel alpha(1C) (Ca(V)1.2) and beta subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the alpha(1C) subunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the alpha(1C) subunit, a domain consisting of amino acid residues 1623-1666 ("targeting domain") in the C terminus of the alpha(1C) subunit has been identified to be important for correct targeting of L-type Ca(2+) channel complexes to the plasma membrane. Although cells expressing the wild-type alpha(1C) and beta(2a) subunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the alpha(1C) subunit that lack the targeting domain with the beta(2a) subunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the alpha(1C) subunit that were downstream of residues 1623-1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the alpha(1C) subunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the alpha(1C) subunit was required for proper plasma membrane localization of the L-type Ca(2+) channels.     

Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel.

The skeletal muscle dihydropyridine receptor/Ca2+ channel is composed of five protein components (alpha 1, alpha 2 delta, beta, and gamma). Only two such components, alpha 1 and alpha 2, have been identified in heart. The present study reports the cloning and expression of a novel beta gene that is expressed in heart, lung, and brain. Coexpression of this beta with a cardiac alpha 1 in Xenopus oocytes causes the following changes in Ca2+ channel activity: it increases peak currents, accelerates activation kinetics, and shifts the current-voltage relationship toward more hyperpolarized potentials. It also increases dihydropyridine binding to alpha 1 in COS cells. These results indicate that the cardiac L-type Ca2+ channel has a similar subunit structure as in skeletal muscle, and provides evidence for the modulatory role of the beta subunit.     

The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.     

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.     

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.     

Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes

Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.     

I-II loop structural determinants in the gating and surface expression of low voltage-activated calcium channels

The intracellular loops that interlink the four transmembrane domains of Ca(2+)- and Na(+)-channels (Ca(v), Na(v)) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I-II loop) in high voltage-activated (HVA) Ca(2+) channels possesses the binding site for Ca(v)beta subunits and plays significant roles in channel function, including trafficking the alpha(1) subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I-II loop of Ca(v)1/Ca(v)2 HVA channels and Ca(v)3 LVA/T-type channels, evidence for a regulatory role of the I-II loop in T-channel function has recently emerged for Ca(v)3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Ca(v)3 channels, we have performed a structure-function analysis of the I-II loop in Ca(v)3.1 and Ca(v)3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Ca(v)3.1 and Ca(v)3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Ca(v)3 channels. In contrast to findings in Ca(v)3.2, deletion of the central region of the I-II loop in Ca(v)3.1 and Ca(v)3.3 yielded a modest increase (+30%) and a reduction (-30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Ca(v)3 function by highlighting the unique role played by the intracellular I-II loop in Ca(v)3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Ca(v)3.3.     

Di-arginine signals and the K-rich domain retain the Ca²⁺ sensor STIM1 in the endoplasmic reticulum

STIM1 is a core component of the store‐operated Ca²⁺‐entry channel involved in Ca²⁺‐signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca²⁺ concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related protein STIM2 functions similar but its physiological role is less well defined. We found that STIM2, but not STIM1, contains a di‐lysine ER‐retention signal. This restricts the function of STIM2 as Ca²⁺ sensor to the ER while STIM1 can reach the plasma membrane. The intracellular distribution of STIM1 is regulated in a cell‐cycle‐dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its lysine‐rich domain and a di‐arginine ER retention signal. Store‐operated Ca²⁺‐entry enhanced ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1s role as multifunctional component in Ca²⁺‐signaling.     

Competitive and synergistic interactions of G protein beta(2) and Ca(2+) channel beta(1b) subunits with Ca(v)2.1 channels, revealed by mammalian two-hybrid and fluorescence resonance energy transfer measurements

Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein betagamma subunits modulate the binding of the Ca2+ channel beta subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel beta and G protein beta subunits on the two separate binding sites, i.e. the loopI-II and the C terminus, and on the full-length P/Q-type alpha12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel beta1b subunit induces a strong fluorescent signal when interacting with the loopI-II but not with the C terminus. In contrast, the G protein beta subunit induces FRET signals on both the C terminus and loopI-II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel beta1b and G protein beta subunits bind to the alpha1 subunit at the same time. Coexpression of the G protein increases the FRET signal between alpha1/beta1b FRET pairs but not for alpha1/beta1b FRET pairs where the C terminus was deleted from the alpha1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel beta and alpha12.1 subunits, which involves the C terminus of the alpha1 subunit and may corresponds to a new conformational state of the channel.     

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.     

Unique modulation of L-type Ca2+ channels by short auxiliary beta1d subunit present in cardiac muscle

Recent studies have identified a growing diversity of splice variants of auxiliary Ca2+ channel Ca(v)beta subunits. The Ca(v)beta(1d) isoform encodes a putative protein composed of the amino-terminal half of the full-length Ca(v)beta(1) isoform and thus lacks the known high-affinity binding site that recognizes the Ca2+ channel alpha1-subunit, the alpha-binding pocket. The present study investigated whether the Ca(v)beta(1d) subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Ca(v)beta subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Ca(v)beta(1d) identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Ca(v)beta(1b) subunit promoted membrane trafficking of the pore-forming alpha1C (Ca(v)1.2)-subunit to the surface membrane, whereas the Ca(v)beta(1d) subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Ca(v)beta(1d) with the alpha1C-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Ca(v)beta(1b) coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Ca(v)beta(1b) or Ca(v)beta(1d) significantly increased mean open probability four- to fivefold relative to the alpha1C-channels alone, but only Ca(v)beta(1b) coexpression increased the number of channels observed per patch. In conclusion, the Ca(v)beta(1d) isoform is expressed in heart and can modulate the gating of L-type Ca2+ channels, but it does not promote membrane trafficking of the channel complex.