A three-way inter-molecular network accounts for the CaVα2δ1-induced functional modulation of the pore-forming CaV1.2 subunit.

L-type CaV1.2 channels are essential for the excitation-contraction coupling in cardiomyocytes and are hetero-oligomers of a pore-forming CaVα1C assembled with CaVß and CaVα2δ1 subunits. A direct interaction between CaVα2δ1 and Asp-181 in the first extracellular loop of CaVα1 reproduces the native properties of the channel. A 3D model of the von Willebrand factor type A (VWA) domain of CaVα2δ1 complexed with the voltage sensor domain of CaVα1C suggests that Ser-261 and Ser-263 residues in the metal ion-dependent adhesion site (MIDAS) motif are determinant in this interaction, but this hypothesis is untested. Here, coimmunoprecipitation assays and patch-clamp experiments of single-substitution variants revealed that CaVα2δ1 Asp-259 and Ser-261 are the two most important residues in regard to protein interactions and modulation of CaV1.2 currents. In contrast, mutating the side chains of CaVα2δ1 Ser-263, Thr-331, and Asp-363 with alanine did not completely prevent channel function. Molecular dynamics simulations indicated that the carboxylate side chain of CaVα2δ1 Asp-259 coordinates the divalent cation that is further stabilized by the oxygen atoms from the hydroxyl side chain of CaVα2δ1 Ser-261 and the carboxylate group of CaVα1C Asp-181. In return, the hydrogen atoms contributed by the side chain of Ser-261 and the main chain of Ser-263 bonded the oxygen atoms of CaV1.2 Asp-181. We propose that CaVα2δ1 Asp-259 promotes Ca2+ binding necessary to produce the conformation of the VWA domain that locks CaVα2δ1 Ser-261 and Ser-263 within atomic distance of CaVα1C Asp-181. This three-way network appears to account for the CaVα2δ1-induced modulation of CaV1.2 currents.

Negatively charged residues in the first extracellular loop of the L-type CaV1.2 channel anchor the interaction with the CaVα2δ1 auxiliary subunit.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes. Co-expression of CaVα2δ1 with CaVß/CaVα1 proteins reconstitutes the functional properties of native L-type currents, but the interacting domains at the CaV1.2/CaVα2δ1 interface are unknown. Here, a homology-based model of CaV1.2 identified protein interfaces between the extracellular domain of CaVα2δ1 and the extracellular loops of the CaVα1 protein in repeats I (IS1S2 and IS5S6), II (IIS5S6), and III (IIIS5S6). Insertion of a 9-residue hemagglutinin epitope in IS1S2, but not in IS5S6 or in IIS5S6, prevented the co-immunoprecipitation of CaV1.2 with CaVα2δ1. IS1S2 contains a cluster of three conserved negatively charged residues Glu-179, Asp-180, and Asp-181 that could contribute to non-bonded interactions with CaVα2δ1. Substitutions of CaV1.2 Asp-181 impaired the co-immunoprecipitation of CaVß/CaV1.2 with CaVα2δ1 and the CaVα2δ1-dependent shift in voltage-dependent activation gating. In contrast, single substitutions in CaV1.2 in neighboring positions in the same loop (179, 180, and 182-184) did not significantly alter the functional up-regulation of CaV1.2 whole-cell currents. However, a negatively charged residue at position 180 was necessary to convey the CaVα2δ1-mediated shift in the activation gating. We also found a more modest contribution from the positively charged Arg-1119 in the extracellular pore region in repeat III of CaV1.2. We conclude that CaV1.2 Asp-181 anchors the physical interaction that facilitates the CaVα2δ1-mediated functional modulation of CaV1.2 currents. By stabilizing the first extracellular loop of CaV1.2, CaVα2δ1 may up-regulate currents by promoting conformations of the voltage sensor that are associated with the channel's open state.

Proteolytic cleavage of the hydrophobic domain in the CaVα2δ1 subunit improves assembly and activity of cardiac CaV1.2 channels.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVß, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.

Inherited Ventricular Arrhythmias: The Role of the Multi-Subunit Structure of the L-Type Calcium Channel Complex.

The normal heartbeat is conditioned by transient increases in the intracellular free Ca2+ concentration. Ca2+ influx in cardiomyocytes is regulated by the activity of the heteromeric L-type voltage-activated CaV1.2 channel. A complex network of interactions between the different proteins forming the ion channel supports the kinetics and the activation gating of the Ca2+ influx. Alterations in the biophysical and biochemical properties or in the biogenesis in any of these proteins can lead to serious disturbances in the cardiac rhythm. The multi-subunit nature of the channel complex is better comprehended by examining the high-resolution three-dimensional structure of the closely related CaV1.1 channel. The architectural map identifies precise interaction loci between the different subunits and paves the way for elucidating the mechanistic basis for the regulation of Ca2+ balance in cardiac myocytes under physiological and pathological conditions.

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

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 Ca(2+) channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 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 CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 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 CaVα2δ1 as well as the CaVα2δ1-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 CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca(2+) channels.

Functional characterization of CaVα2δ mutations associated with sudden cardiac death.

L-type Ca(2+) channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVß and CaVα2δ subunits. CaVα2δ increases the peak current density and improves the voltage-dependent activation gating of CaV1.2 channels without increasing the surface expression of the CaVα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for CaVα2δ), 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 CaV1.2 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 CaVα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of CaVα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the CaVα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of CaVα2δ D550Y, CaVα2δ 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 CaVα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased CaV1.2 currents as compared with results obtained with CaVα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca(2+) currents through a defect in the cell surface trafficking of CaVα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of CaV1.2 currents by CaVα2δ.