Effect of Cavβ Subunits on Structural Organization of Cav1.2 Calcium Channels

Background

Voltage-gated Ca_v1.2 calcium channels play a crucial role in Ca²⁺ signaling. The pore-forming α_1C subunit is regulated by accessory Ca_vβ subunits, cytoplasmic proteins of various size encoded by four different genes (Ca_vβ₁ - β₄) and expressed in a tissue-specific manner.

Methods and Results

Here we investigated the effect of three major Ca_vβ types, β_1b, β_2d and β₃, on the structure of Ca_v1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Ca_v1.2 to form clusters depends on the type of the Ca_vβ subunit present. The highest density of Ca_v1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac β_1b present. Ca_v1.2 channels containing β₃, the predominant Ca_vβ subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between α_1C and Ca_vβ in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Ca_v1.2 channels in the plasma membrane depends on the Ca_vβ type. The presence of different Ca_vβ subunits does not result in significant differences in the intramolecular distance between the termini of α_1C, but significantly affects the distance between the termini of neighbor α_1C subunits, which varies from 67 Å with β_1b to 79 Å with β₃.

Conclusions

Thus, our results show that the structural organization of Ca_v1.2 channels in the plasma membrane depends on the type of Ca_vβ subunits present.     

Hypoxia-inducible factor-1α regulates the expression of L-type voltage-dependent Ca2+ channels in PC12 cells under hypoxia

Hypoxia is an important factor in regulation of cell behavior both under physiological and pathological conditions. The mechanisms of hypoxia-induced cell death have not been completely elucidated yet. It is well known that Ca²⁺ is critically related to cell survival. Hypoxia-inducible factor-1α (HIF-1α) is a core regulatory factor during hypoxia, and L-type voltage-dependent Ca²⁺ channels (L-VDCCs) have been reported to play a critical role in cell survival. This study was conducted to explore the relationship between L-VDCC expression and HIF-1α regulation in PC12 cells under hypoxia. PC12 cells were treated at 20 or 3 % O₂ to observe its proliferation and the intracellular calcium concentration. Then, we detected the protein expression of HIF-1α and L-VDCCs subtypes, Ca_v1.2 and Ca_v1.3. At last, to verify the relationship between HIF-1α and Ca_v1.2 and Ca_v1.3, we got the expression of Ca_v1.2 and Ca_v1.3 with Western blot and luciferase report gene assays after PC12 cells were treated by echinomycin, which is an HIF-1α inhibitor. Compared with 20 % O₂ (normoxia), 3 % O₂ (hypoxia) inhibited cell proliferation, increased the intracellular calcium concentration, and induced protein expression of HIF-1α. The protein expression of two L-VDCCs subtypes expressed in the nervous system, Ca_v1.2 and Ca_v1.3, was upregulated by hypoxia and reduced dose dependently by treatment with echinomycin, a HIF-1α inhibitor. Luciferase report gene assays showed that the expression of Ca_v1.2 and Ca_v1.3 genes was augmented under 3 % O₂. However, echinomycin only slightly and dose dependently decreased expression of the Ca_v1.2 gene, but not that of the Ca_v1.3 gene. These data indicated that Ca_v1.2 might be regulated by HIF-1α as one of its downstream target genes and involved in regulation of PC12 cells death under hypoxia.     

Characterization of Cav1.4 Complexes (α11.4, β2, and α2δ4) in HEK293T Cells and in the Retina

In photoreceptor synaptic terminals, voltage-gated Ca_v1.4 channels mediate Ca²⁺ signals required for transmission of visual stimuli. Like other high voltage-activated Ca_v channels, Ca_v1.4 channels are composed of a main pore-forming Ca_v1.4 α₁ subunit and auxiliary β and α₂δ subunits. Of the four distinct classes of β and α₂δ, β₂ and α₂δ₄ are thought to co-assemble with Ca_v1.4 α₁ subunits in photoreceptors. However, an understanding of the functional properties of this combination of Ca_v subunits is lacking. Here, we provide evidence that Ca_v1.4 α₁, β₂, and α₂δ₄ contribute to Ca_v1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of β₂, named here β_2X13, which, along with β_2a, is present in photoreceptor terminals. Ca_v1.4 α_1, β₂, and α₂δ₄ were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Ca_v1.4 α₁ + β_2X13) containing α₂δ₄ exhibited weaker voltage-dependent activation than those with α₂δ₁. Moreover, compared with channels (Ca_v1.4 α₁ + α₂δ₄) with β_2a, β_2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Ca_v1.4 because it was not seen for Ca_v1.2 channels. Our results provide the first detailed functional analysis of the Ca_v1.4 subunits that form native photoreceptor Ca_v1.4 channels and indicate potential heterogeneity in these channels conferred by β_2a and β_2X13 variants.     

BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca²⁺-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca²⁺ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Ca_vβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α₁ interaction domain–binding pocket in Ca_vβ and interferes with the association between Ca_vβ and Ca_vα1. In the absence of domain I binding, BARP can form a ternary complex with Ca_vα1 and Ca_vβ via domain II. BARP does not affect cell surface expression of Ca_vα1 but inhibits Ca²⁺ channel activity at the plasma membrane, resulting in the inhibition of Ca²⁺-evoked exocytosis. Thus, BARP can modulate the localization of Ca_vβ and its association with the Ca_vα1 subunit to negatively regulate VGCC activity.     

Gene Splicing of an Invertebrate Beta Subunit (LCavβ) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels

The accessory beta subunit (Ca_vβ) of calcium channels first appear in the same genome as Ca_v1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Ca_vβ subunits (β₁, β₂, β₃, β₄) which associate with four Ca_v1 channel isoforms (Ca_v1.1 to Ca_v1.4) and three Ca_v2 channel isoforms (Ca_v2.1 to Ca_v2.3). Here we assess the fundamentally-shared features of the Ca_vβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCa_v1, LCa_v2, and LCa_vβ). Invertebrate Ca_vβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Ca_v2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCa_vβ subunit. LCa_vβ will also slow the inactivation kinetics of LCa_v3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Ca_vβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa_vβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Ca_v2.2 and Ca_vβ₃ subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCa_v2 nor LCa_vβ) appears to be compatible with this observed property.     

Calretinin Regulates Ca2+-dependent Inactivation and Facilitation of Cav2.1 Ca2+ Channels through a Direct Interaction with the α12.1 Subunit

Voltage-gated Ca_v2.1 Ca²⁺ channels undergo dual modulation by Ca²⁺, Ca²⁺-dependent inactivation (CDI), and Ca²⁺-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca²⁺-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α₁2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α₁2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca²⁺-dependent. α₁2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca_v2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca_v2.1 CDI and increased CDF. CR binding to α₁2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α₁2.1 with that from the Ca_v1.2 α₁ subunit (α₁1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α₁2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca_v2.1, which may have major consequences for Ca²⁺ signaling and neuronal excitability.     

Calcium Channel α2δ1 Proteins Mediate Trigeminal Neuropathic Pain States Associated with Aberrant Excitatory Synaptogenesis

To investigate a potential mechanism underlying trigeminal nerve injury-induced orofacial hypersensitivity, we used a rat model of chronic constriction injury to the infraorbital nerve (CCI-ION) to study whether CCI-ION caused calcium channel α₂δ₁ (Ca_vα₂δ₁) protein dysregulation in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 cervical dorsal spinal cord (Vc/C2). Furthermore, we studied whether this neuroplasticity contributed to spinal neuron sensitization and neuropathic pain states. CCI-ION caused orofacial hypersensitivity that correlated with Ca_vα₂δ₁ up-regulation in trigeminal ganglion neurons and Vc/C2. Blocking Ca_vα₂δ₁ with gabapentin, a ligand for the Ca_vα₂δ₁ proteins, or Ca_vα₂δ₁ antisense oligodeoxynucleotides led to a reversal of orofacial hypersensitivity, supporting an important role of Ca_vα₂δ₁ in orofacial pain processing. Importantly, increased Ca_vα₂δ₁ in Vc/C2 superficial dorsal horn was associated with increased excitatory synaptogenesis and increased frequency, but not the amplitude, of miniature excitatory postsynaptic currents in dorsal horn neurons that could be blocked by gabapentin. Thus, CCI-ION-induced Ca_vα₂δ₁ up-regulation may contribute to orofacial neuropathic pain states through abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release in Vc/C2.     

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

Background

The Ca_vβ subunits of high voltage-activated Ca²⁺ channels control the trafficking and biophysical properties of the α₁ subunit. The Ca_vβ-α₁ interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that βs regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the α-interaction domain (AID) be a rigid structure.

Methodology/Principal Findings

The present study tests this hypothesis by altering the flexibility and orientation of this region in α₁2.2, then testing for Ca_vβ regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished β2a and β3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of β2a to produce non-inactivating currents. Orientation of Ca_vβ with respect to α₁2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca_vβ subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca_vβ subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms β-sheet. The orientation of β with respect to α was confirmed by the bimolecular fluorescence complementation assay.

Conclusions/Significance

These results show that the orientation of the Ca_vβ subunit relative to the α₁2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca_vβ to regulate channel activity.     

Assembly of a Ca2+-dependent BK channel signaling complex by binding to beta2 adrenergic receptor

Large-conductance voltage and Ca²⁺-activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of β-adrenergic receptors and protein kinase A (PKA_c) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the β2-adrenergic receptor (β2AR) with the pore forming α subunit of BKCa and an A-kinase-anchoring protein (AKAP79/150) for β2 agonist regulation. β2AR can simultaneously interact with both BKCa and L-type Ca²⁺ channels (Ca_v1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca²⁺- and phosphorylation-dependent modulation of BKCa current. Our findings reveal a novel function for G protein-coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate β-adrenergic regulation of membrane excitability.     

Role of PKC and CaV1.2 in Detrusor Overactivity in a Model of Obesity Associated with Insulin Resistance in Mice

Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM) in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC) and Ca_v1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Ca_v1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001–100 µM), α,β-methylene ATP (1–10 µM), KCl (1–300 mM), extracellular Ca²⁺ (0.01–100 mM) and phorbol-12,13-dibutyrate (PDBu; 0.001–3 µM) all produced greater DSM contractions in obese mice, which were fully reversed by the Ca_v1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM) also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Ca_v1.2 channel protein expression was not modified in any experimental group. Our findings show that Ca_v1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.     

Molecular Determinants of the CaVβ-induced Plasma Membrane Targeting of the CaV1.2 Channel

Ca_Vβ subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca_V1 and Ca_V2 α1 subunits. High affinity Ca_Vβ binding onto the so-called α interaction domain of the I-II linker of the Ca_Vα1 subunit is required for Ca_Vβ modulation of HVA channel gating. It has been suggested, however, that Ca_Vβ-mediated plasma membrane targeting could be uncoupled from Ca_Vβ-mediated modulation of channel gating. In addition to Ca_Vβ, Ca_Vα2δ and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca_Vα2δ caused a 5-fold stimulation of the whole cell currents measured with Ca_V1.2 and Ca_Vβ3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca_V1.2, extracellularly tagged Ca_V1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca_V1.2 with either Ca_Vα2δ, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca_V1.2 subunits. In contrast, co-expression with Ca_Vβ3 stimulated plasma membrane expression of Ca_V1.2 by a 10-fold factor. Mutations within the α interaction domain of Ca_V1.2 or within the nucleotide kinase domain of Ca_Vβ3 disrupted the Ca_Vβ3-induced plasma membrane targeting of Ca_V1.2. Altogether, these data support a model where high affinity binding of Ca_Vβ to the I-II linker of Ca_Vα1 largely accounts for Ca_Vβ-induced plasma membrane targeting of Ca_V1.2.     

Functional Characterization of CaVα2δ Mutations Associated with Sudden Cardiac Death

L-type Ca²⁺ channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming Ca_Vα1 with the auxiliary Ca_Vβ and Ca_Vα2δ subunits. Ca_Vα2δ increases the peak current density and improves the voltage-dependent activation gating of Ca_V1.2 channels without increasing the surface expression of the Ca_Vα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for Ca_Vα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type Ca_V1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the Ca_Vα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of Ca_Vα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the Ca_Vα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of Ca_Vα2δ D550Y, Ca_Vα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30–33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of Ca_Vα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased Ca_V1.2 currents as compared with results obtained with Ca_Vα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca²⁺ currents through a defect in the cell surface trafficking of Ca_Vα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of Ca_V1.2 currents by Ca_Vα2δ.     

A Single Immunoglobulin-like Domain of the Human Neural Cell Adhesion Molecule L1 Supports Adhesion by Multiple Vascular and Platelet Integrins

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃. The interaction between L1-Ig6 and α_IIbβ₃ was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with α_vβ₃ and α_vβ₁ supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃. Whereas β₃ integrin binding to L1-Ig6 was evident in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, a corresponding interaction with the β₁ integrins was only observed in the presence of Mn²⁺. Furthermore, such Mn²⁺-dependent binding by α₅β₁ and α_vβ₁ was significantly inhibited by exogenous Ca²⁺. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that α_vβ₃ or active α_IIbβ₃ > α_vβ₁ > α₅β₁. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.     

Foot-and-Mouth Disease Virus Virulent for Cattle Utilizes the Integrin αvβ3 as Its Receptor

Adsorption and plaque formation of foot-and-mouth disease virus (FMDV) serotype A₁₂ are inhibited by antibodies to the integrin α_vβ₃ (A. Berinstein et al., J. Virol. 69:2664–2666, 1995). A human cell line, K562, which does not normally express α_vβ₃ cannot replicate this serotype unless cells are transfected with cDNAs encoding this integrin (K562-α_vβ₃ cells). In contrast, we found that a tissue culture-propagated FMDV, type O₁BFS, was able to replicate in nontransfected K562 cells, and replication was not inhibited by antibodies to the endogenously expressed integrin α₅β₁. A recent report indicating that cell surface heparan sulfate (HS) was required for efficient infection of type O₁ (T. Jackson et al., J. Virol. 70:5282–5287, 1996) led us to examine the role of HS and α_vβ₃ in FMDV infection. We transfected normal CHO cells, which express HS but not α_vβ₃, and two HS-deficient CHO cell lines with cDNAs encoding human α_vβ₃, producing a panel of cells that expressed one or both receptors. In these cells, type A₁₂ replication was dependent on expression of α_vβ₃, whereas type O₁BFS replicated to high titer in normal CHO cells but could not replicate in HS-deficient cells even when they expressed α_vβ₃. We have also analyzed two genetically engineered variants of type O₁Campos, vCRM4, which has greatly reduced virulence in cattle and can bind to heparin-Sepharose columns, and vCRM8, which is highly virulent in cattle and cannot bind to heparin-Sepharose. vCRM4 replicated in wild-type K562 cells and normal, nontransfected CHO (HS⁺ α_vβ_3⁻) cells, whereas vCRM8 replicated only in K562 and CHO cells transfected with α_vβ₃ cDNAs. A similar result was also obtained in assays using a vCRM4 virus with an engineered RGD→KGE mutation. These results indicate that virulent FMDV utilizes the α_vβ₃ integrin as a primary receptor for infection and that adaptation of type O₁ virus to cell culture results in the ability of the virus to utilize HS as a receptor and a concomitant loss of virulence.     

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca²⁺ channel complexes. Ca_Vα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca_V1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca_Vα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca_Vα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca_Vα2δ1-mediated increase in the peak current density and voltage-dependent gating of Ca_V1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca_Vα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca_Vα2δ1 as well as the Ca_Vα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca_Vα2δ1, and furthermore that N-glycosylation of Ca_Vα2δ1 is essential to produce functional L-type Ca²⁺ channels.     

Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells

Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Ca_v). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Ca_v opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet β-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Ca_vα1 pore-forming subunits of R-type Ca_v2.3 and to lesser extent L-type Ca_vs, while confirming the preferential interactions between Syn-1A with L-type (Ca_v1.2, Ca_v1.3) Ca_vs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Ca_v2.3, whereas Syn-1A prefers L-type Ca_vs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Ca_v channels. Syn-3 KD enhanced Ca²⁺ currents by 46% attributed mostly to R- and L-type Ca_vs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Ca_v activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Ca_v activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Ca_vs, but preferring Ca_v2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS.