Functional Characterization of CaVα2δ Mutations Associated with Sudden Cardiac Death*

Background: Missense mutations in CaVα2δ1, an auxiliary subunit of cardiac L-type CaV1.2 channels, are associated with arrhythmias. Results: The reduction in the cell surface density of CaVα2δ1 D550Y/Q917H was sufficient to impair CaV1.2 currents. Conclusion: Defects in the cell surface trafficking of CaVα2δ1 mutants down-regulate L-type currents. Significance: CACNA2D1 genetic variants may trigger arrhythmias by reducing L-type Ca2+ currents. L-type Ca2+ channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVβ 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 Ca2+ 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.

Polymorphic ventricular tachycardia is one of the leading causes of sudden cardiac death in children and young adults (1). These cardiac arrhythmias, which are reported in the absence of structural heart defects, coronary artery disease, or heart failure, are detected in a noninvasive fashion by measuring changes in the QT 3 interval on the electrocardiogram. Either excessive prolongation (LQT) or shortening of the QT (SQT) intervals are associated with an increased risk of sudden cardiac death (2). Inherited Mendelian long QT syndrome (LQTS) and short QT syndrome (SQTS) originate from mutations in genes encoding ion channels or channel-interacting proteins (3). Genome-wide linkage studies of families with LQTS have reported 13 LQTS susceptibility genes with 75% of LQTS cases stemming from mutations in KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3) (4). Only recently have mutations of genes coding for subunits forming the L-type Ca 2ϩ channel been linked to inherited arrhythmogenic diseases caused by LQTS or SQTS (5). The molecular mechanism underlying the cardiac dysfunction remains unknown in many cases.
The CACNA1C gene encodes the L-type Ca V 1.2 channel that carries the vast majority of the L-type calcium current in the adult heart (6,7) which in turn initiates the coordinated contraction of the cardiac ventricles (8). Its unique role is substantiated by the observation that homozygous knock-out of the CACNA1C gene is lethal (9,10). In addition to initiating the coordinated contraction of the cardiac ventricles, Ca V 1.2 channels are critical to the heart's normal rhythmic activity (8). During the cardiac action potential, Ca 2ϩ enters the cell through the voltage-dependent L-type Ca V 1.2 generating an inward Ca 2ϩ current that contributes to the plateau phase of the action potential (11). The kinetic properties of this channel must be properly timed so that depolarization and contraction are synchronized during the systolic-diastolic cycle of the heart. Even a slight disruption of Ca 2ϩ cycling can have a profound impact on action-potential duration and trigger early after depolarizations, ultimately cumulating in lethal * This work was supported by Operating Grant 130256 from the Canadian Institutes of Health Research and a grant from the Canadian Heart and Stroke Foundation (to L. P.). 1 Both authors contributed equally to this work. 2  arrhythmias (5,12), including torsade de pointes and ventricular fibrillation (2). Prolonged inward Ca 2ϩ current during the plateau phase of the cardiac action potential leads to delays in ventricular myocyte repolarization, a subsequent prolonged QT repolarization interval on the electrocardiogram and a highly arrhythmogenic and potentially lethal substrate. On the other end of the spectrum, any significant decrease in inward currents or significant increase in outward currents may lead to lethal arrhythmias associated with shorter than normal QT repolarization intervals (3). The L-type Ca V 1.2 channel belongs to the molecular family of high voltage activated Ca V channels. High voltage-activated Ca V 1.2 channels are hetero-oligomers formed by the main pore-forming Ca V ␣1 subunit in a complex with the cytoplasmic Ca V ␤ auxiliary subunit, the EF-hand protein calmodulin constitutively bound to the C terminus of Ca V ␣1, and the mostly extracellular Ca V ␣2␦ subunit (13)(14)(15)(16)(17)(18). The full complement of auxiliary subunits is required to produce high voltage-activated Ca V 1.2 channels with the properties of the native channels. Ca V ␤ promotes the cell surface density of Ca V 1.2 channels (19) in part by preventing its degradation by the ubiquitin/proteasome system (20). Co-expression of Ca V ␣2␦ subunit with Ca V ␤-bound Ca V ␣1 increases peak current density and hyperpolarizes the voltage of activation of the L-type Ca V 1.2 (19,(21)(22)(23).
Gain-of-function mutations in the pore-forming Ca V ␣1 subunit of the L-type Ca 2ϩ channel lead to the highly arrhythmogenic Timothy syndrome. Timothy syndromes 1 and 2 are rare variants of the long QT syndrome (LQT8) (24,25) characterized by extreme QT-prolongation and gain-of-function mutations in the pore-forming subunit of Ca V 1.2 (24 -28). Mutations in the auxiliary subunits forming the L-type Ca V 1.2 channel have also been identified among probands diagnosed with Brugada syndrome, idiopathic ventricular fibrillation, and early repolarization syndrome associated with short QT interval (QTc Յ360 ms) (29 -31). According to these studies, the shorter QT interval might result from loss-of-function mutations in the Ca V genes coding for Ca V ␣1 (CACNA1C), Ca V ␤ (CACNB2), or Ca V ␣2␦ (CACNA2D1). Missense mutations D550Y, S709N, S755T, Q917H, and S956T in the Ca V ␣2␦1 protein have been associated with congenital arrhythmias (referred to as short QT syndrome 6 (SQT6) (29 -31)), but the molecular mechanism(s) underlying the change in function remains to be established (32).
In this work, we have characterized the Ca V ␣2␦ mutations associated with arrhythmogenic activity in regard to their functional impact on the cardiac L-type Ca V 1.2 currents. Using a fluorescence-labeled and extracellularly tagged Ca V ␣2␦ subunit, we show here that missense mutations D550Y, S709N and D550Y/Q917H significantly altered the cell surface density of Ca V ␣2␦. Decreasing the cell surface density of Ca V ␣2␦ D550Y/Q917H was found to profoundly decrease L-type peak current densities as compared with currents measured with Ca V ␣2␦ wild type (WT). Altogether, our data support a model where Ca V ␣2␦ modulates channel function without altering the trafficking of Ca V 1.2 and establishes the cell surface density of Ca V ␣2␦ relative to the pore-forming Ca V ␣1 as the single most important determinant in the stimulation of Ca V 1.2 currents by Ca V ␣2␦.

EXPERIMENTAL PROCEDURES
Recombinant DNA Techniques-The rabbit Ca V 1.2 (Gen-Bank TM X15539), the rat Ca V ␤3 (GenBank TM M88751) (33), and the rat brain Ca V ␣2␦1 (GenBank TM NM_012919) (34) were subcloned in commercial vectors under the control of the CMV promoter as described elsewhere (19,35). The primary sequence of the rat brain Ca V ␣2␦1 clone (1091 residues) is 96% identical to the predicted sequence of the human clone NM_000722 (1091 residues). All residues mutated in the following experiments are conserved between species. The first series of experiments (Figs. 1 and 2) was conducted with a tagged version of Ca V 1.2 whereby the hemagglutinin (HA) epitope (YPYDVPDYA) was inserted in the extracytoplasmic loop of Ca V 1.2 in the S5-S6 linker of domain II between residues 710 and 711, whereas the untagged version of Ca V 1.2 was used in the experiments shown in Figs. 3-11. The rat Ca V ␣2␦1 was subcloned in the pmCherry-N1 vector (Cederlane, Burlington, Ontario, Canada) between the SacI and SalI sites. In this construct, the hemagglutinin (HA) epitope (YPYDVPDYA) was inserted in the extracellular domain of Ca V ␣2. More than 10 different insertion sites were tested in Ca V ␣2, but only one was found to be functional and fluorescently labeled with a FITClabeled anti-HA (fluorescein isothiocyanate) (results not shown). The insertion site was identified at position Arg-676 in the extracellular domain. Cell surface and total density of Ca V ␣2␦ were evaluated by two-color flow cytometry assays using the pmCherry-Ca V ␣2␦1-HA construct. Unless specified otherwise, the flow cytometry experiments and the patch clamp recordings were systematically conducted with this pmCherry-Ca V ␣2␦-1-HA construct.
Site-directed Mutagenesis-All mutants were produced with the Q5 site-directed mutagenesis kit (New England Biolabs, Whitby, Ontario, Canada) according to the manufacturer's instructions. Briefly, amino acid substitutions were performed by nonoverlapping desalted primers. Substitutions are created by incorporating the desired mutation in the center of the forward primer, and the reverse primer is designed so that the 5Ј ends of the two primers anneal back-to-back. Following the PCR, a kinase/ligase/DpnI enzyme mixture was added to the amplified DNA for circularization and template removal before transformation into high efficiency DH5␣-competent Escherichia coli. Constructs were verified by automated doublestranded sequence analysis (Genomics Platform, IRIC, Université de Montréal, Quebec, Canada). Protein expression of all constructs was confirmed by Western blotting in total cell lysates as described previously (19).
Cell Culture and Transfections-HEK293T or HEKT cells were grown in Dulbecco's high glucose minimum essential medium (DMEM-HG) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin at 37°C under 5% CO 2 atmosphere as described elsewhere (19). RT-PCR conducted in these cells failed to highlight the presence of Ca V ␤ and Ca V ␣2␦ auxiliary subunits (36). All experiments described herein were conducted in HEKT cells stably transfected with Ca V ␤3, as this isoform was best at promoting the cell surface density of Ca V 1.2 (19). The use of stable cells limited the problems linked to protein overexpression. Unless specified otherwise, stable Ca V ␤3 cells (90% confluence) were transiently transfected with similar amounts of DNA (4 g each for a total of 8 g total per 10 6 cells) as follows: Ca V 1.2 WT-HA (Figs. 1 and 2), Ca V 1.2 WT (Figs. [3][4][5][6][7][8][9][10][11], Ca V ␣2␦1 WT, Ca V ␣2␦1-HA, or empty vector in 10 l of Lipofectamine 2000 (Invitrogen) using a DNA/lipid ratio of 1:2.5 as described elsewhere (19). In some experiments, the amount of DNA for Ca V ␣2␦ was decreased in the 0.2-4.0-g range to decrease the Ca V ␣2␦/Ca V 1.2 expression ratio, but the total DNA amount was kept constant at 8 g by using a mock vector.
Flow Cytometry Experiments-Flow cytometry experiments were conducted as described elsewhere (19,35). To determine the cell surface expression level of the tagged proteins, cells were harvested 24 h after transfection, washed in a PBS 1ϫ buffer, and stained with the FITC-labeled anti-HA epitope tag antibody at 5 g/ml (Clone HA-7, Sigma) at 4°C for 30 min. To determine the density of both intracellular and extracellular expression of the tagged proteins, cells were fixed and permeabilized using BD Cytofix/Cytoperm TM fixation/permeabilization solution kit (BD Biosciences, 554714) according to the manufacturer's instructions. Briefly, cells were washed with the 1ϫ BD Perm/Wash buffer containing both FBS and saponin and stained 30 min at 4°C with the FITC-labeled anti-HA epitope tag antibody. A maximum of 10,000 cells were counted using a FACSAria III special order research product flow cytometer (BD Biosciences) at the flow cytometry facility located at the Department of Microbiology, Université de Montréal. Relative expression of Ca V ␣2␦ was calculated based on ⌬mean fluorescence intensity (⌬MFI) for each fluorophore (mCherry or FITC) rather than computing the number of fluorescent cells (see below) (19). Three control conditions were always carried out for each series of experiments as follows: (a) untransfected HEKT cells with the murine IgG1-FITC isotype control (5 g/ml); (b) transfected HEKT cells without the anti-HA FITC antibody but with the murine IgG1-FITC isotype control (5 g/ml); (c) HEKT cells transfected with the wild-type HA-tagged construct. No fluorescence was detected with the IgG1-FITC isotype control murine (5 g/ml) nor in untransfected cells with the anti-HA FITC conjugated antibody (5 g/ml) (data not shown). The fluorescence intensity of each construct transfected in any given condition was measured using six distinct cell dishes for an average of 60,000 cells for each condition. Flow cytometry experiments performed at 36 h to mimic as closely as possible the experimental conditions used for the patch clamp experiments (see below), yielded quantitatively similar results as experiments performed at 24 h (data not shown).
Quantification of Cell Surface Expression-Flow cytometry data were analyzed, and figures were produced using the FlowJo software, version 10 (TreeStar, Ashland, OR). Dead cells were excluded based on forward scatter/side scatter profiles. The FITC-positive cells gate (P2) and the FITC negative cells gate (P3) were set manually. The FITC fluorescence intensity within the region delineated by the P2 and P3 gate was displayed as cell count versus FITC fluorescence intensity (histograms in Figs. 4, 5, and 8). The ⌬MFI for FITC was calculated by subtracting the FITC fluorescence density of the FITC-negative cells (P3) from the fluorescence density of the FITC-positive cells (P2). ⌬MFI was used as an index of the cell surface density of HA-tagged proteins (either HA-Ca V 1.2 or pmCherry-Ca V ␣2␦1-HA) in intact nonpermeabilized cells or the total expression (cell surface and intracellular protein density) of HA-tagged proteins in permeabilized cells. The two HA-tagged proteins were never expressed together. Because cellular autofluorescence levels are altered by the change in the cell medium, the actual ⌬MFI values in arbitrary units cannot be compared between intact nonpermeabilized and permeabilized cells. In Figs. 5 and 9, ⌬MFI were pooled and normalized to the average value obtained for the pmCherry-Ca V ␣2␦1-HA WT construct that was expressed under the same conditions and quantified under the same experimental conditions (see above).
Patch Clamp Experiments in HEKT Cells-Whole-cell voltage clamp recordings were performed on isolated cells 30 -38 h after transfection using the methods described above in the presence of the peGFP vector (0.2 g) as a control for transfection. In all cases, the experiments were carried out under optimal transfection conditions after assessment of the mCherry fluorescence of the mCherry-Ca V ␣2␦ constructs. Patch clamp experiments were carried out with the Axopatch 200-B amplifier (Molecular Devices, Union City, CA). Electrodes were filled with a solution containing (in mM) 140 CsCl, 0.6 NaGTP, 3 MgATP, 10 EGTA, 10 HEPES, titrated to pH 7.3 with NaOH. Pipette resistance ranged from 2 to 4 megohms. Cells were bathed in a modified Earle's saline solution (in mM) as follows: 135 NaCl, 20 tetraethylammonium chloride, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, titrated to pH 7.3 with KOH. PClamp software Clampex 10.2 coupled to a Digidata 1440A acquisition system (Molecular Devices) was used for on-line data acquisition and analysis. Pipette and cell capacitance cancellation and series resistance compensation were applied (up to 80%) using the cancellation feature of the amplifier. Cellular capacitance was estimated by measuring the time constant of current decay evoked by a 10-mV depolarizing pulse applied to the cell from a holding potential of Ϫ100 mV. A series of 450-ms voltage pulses were applied from a holding potential of Ϫ100 mV at a frequency of 0.2 Hz, from Ϫ60 to ϩ70 mV at 5-mV intervals. Unless stated otherwise, data were sampled at 5 kHz and filtered at 1 kHz. Experiments were performed at room temperature (20 -22°C). Activation parameters were estimated from the peak I-V curves obtained for each channel combination and are reported as the mean of individual measurements Ϯ S.E. as described elsewhere (37). Briefly, the I-V relationships were normalized to the maximum amplitude and were fitted to a Boltzmann equation with E 0.5, act being the mid-potential of activation. The free energy of activation was calculated using the mid-activation potential shown in Equation 1, where z is the effective charge displacement during activation, and F is the Faraday constant (38). The r100 ratio is defined as the ratio of peak whole-cell currents remaining after a depolarizing pulse of 100 ms (I 100 ms /I peak ) and was used as an indicator of the inactivation kinetics.
Unless specified otherwise (as in Fig. 1), patch clamp experiments were performed with the untagged version of pCMV-Ca V 1.2 WT transfected with pmCherry-Ca V ␣2␦1-HA WT or mutant in stable Ca V ␤3 cells. Each novel pmCherry-Ca V ␣2␦1-HA mutant was always tested alongside the control condition (pCMV-Ca V 1.2 WT ϩ pmCherry-Ca V ␣2␦1-HA WT in stable Ca V ␤3 cells) to assess for internal consistency. Experiments performed under the same conditions yielded peak current densities Ϯ 20% between samples and between series of experiments. All experiments were pooled and biophysical properties are reported in Table 1.
Statistics-Results were expressed as mean Ϯ S.E. Tests of significance were carried out using the unpaired analysis of variance test embedded in the Origin 7.0 analysis software (OriginLab Corp., Northampton, MA). Data were considered statistically significant at p Ͻ 0.05.

RESULTS
HA-tagged Ca V ␣2␦ Stimulates Ca V 1.2 Currents-Co-expression of Ca V 1.2 and Ca V 2.2 with the auxiliary Ca V ␣2␦ subunit was shown to stimulate whole-cell currents (19,39). Functional modulation of Ca V 1.2 by the various Ca V ␣2␦ constructs was studied after recombinant expression in HEKT cell stably transfected with Ca V ␤3 (Fig. 1A) as explained earlier (19). The immunoreactivity and the integrity of the constructs were verified by Western blotting (data not shown). As shown in Fig. 1A, . Whole-cell current traces were recorded in the presence of 2 mM Ca 2ϩ from a holding potential of Ϫ100 mV. Time scale is 100 ms throughout. Unless specified otherwise, the current density scale is 10 pA/pF. B, peak current densities increased from Ϫ5 Ϯ 1 pA/pF (n ϭ 26) for Ca V 1.2 WT/Ca V ␤3 with an empty pmCherry vector to Ϫ56 Ϯ 3 pA/pF (n ϭ 23) in the presence of Ca V 1.2 WT/Ca V ␤3 with pmCherry-Ca V ␣2␦1 WT. Peak current densities measured in the presence of the following combinations were not statistically significantly different: , a significant Ϫ15 mV shift in the activation potential. The number in parentheses refers to the number of independent experiments. C, histogram is reporting the distribution of the individual ⌬G act values estimated in kilocalories/mol for each experimental condition (see "Experimental Procedures" for details). The ⌬G act values for Ca V 1.2 WT/Ca V ␤3 with pmCherry-Ca V ␣2␦1-HA were best fitted by a Gaussian equation with a ϭ 0.36 centered around Ϫ0.86 kcal mol Ϫ1 . As seen, the distribution of the ⌬G act values measured in the absence of Ca V ␣2␦1 subunit appeared to be flatter (varying from Ϫ0.1 to ϩ 1.5 kcal mol Ϫ1 ) and could not be properly fitted by a Gaussian equation.
whole-cell currents, recorded in the presence of a saline solution containing a physiological concentration of Ca 2ϩ (2 mM), were significantly larger when measured in the presence of the Ca V ␣2␦ confirming that Ca V ␣2␦ stimulates whole-cell currents of Ca V 1.2/Ca V ␤3 (19). Peak current densities increased from Ϫ5 Ϯ 1 pA/pF (n ϭ 26) (no insert in the pmCherry vector) to Ϫ56 Ϯ 3 pA/pF (n ϭ 23) in the presence of Ca V ␣2␦ WT (Fig.  1B). The increase in peak current densities was associated with a Ϫ15-mV leftward shift in the activation potential of Ca V 1.2 from E 0.5, act ϭ 5 Ϯ 2 mV (n ϭ 26) (no Ca V ␣2␦) to E 0.5, act ϭ Ϫ10.1 Ϯ 0.5 mV (n ϭ 23) (with Ca V ␣2␦). Whole-cell currents recorded with untagged Ca V 1.2 WT ϩ mCherry Ca V ␣2␦-HA or with HA-tagged Ca V 1.2 ϩ mCherry Ca V ␣2␦-HA were not significantly different from currents recorded with untagged Ca V 1.2 WT ϩ mCherry Ca V ␣2␦ WT. This result validates the constructions and confirms that the HA epitopes inserted in extracellular loops of both Ca V ␣2␦ and Ca V 1.2 are not occlud-ing each other. The free energy of activation (⌬G act ) measured in the presence of Ca V ␣2␦ was well described by a Gaussian distribution centered around Ϫ0.86 kcal mol Ϫ1 (Fig. 1C), whereas the ⌬G act measured in the absence of Ca V ␣2␦ displayed a broader distribution centered at a value close to Ϸ0 kcal mol Ϫ1 . These results are compatible with a model whereby Ca V ␣2␦ stimulates peak current density by setting Ca V 1.2 channels in a conformational state very close to the open state (40).
Ca V ␣2␦ Improves Total but Not Surface Expression of Ca V 1.2-To evaluate the impact of Ca V ␣2␦ on the protein expression of Ca V 1.2, the HA-tagged version of Ca V 1.2 was expressed in HEKT cells and in stable Ca V ␤3 cells in the presence or absence of Ca V ␣2␦ (Fig. 2). Flow cytometry assays were carried out in the presence of the FITC-conjugated anti-HA in intact and in permeabilized cells. Control experiments carried out with a pCMV-Ca V 1.2 control construct that was not HA- ⌬MFI was used as an index of the cell surface density of Ca V 1.2 in intact nonpermeabilized cells or the total expression (cell surface and intracellular protein density) of Ca V 1.2 in permeabilized cells. As seen, the surface expression of Ca V 1.2 was not significantly improved in the presence of Ca V ␣2␦1 in contrast to the robust stimulation observed with Ca V ␤3. The total protein expression of Ca V 1.2 was improved after co-expression with Ca V ␣2␦1, but stronger stimulation was observed in the combined presence of Ca V ␤3 with Ca V ␣2␦1. C, predicted secondary structure of the HA-tagged Ca V 1.2 construct used in the figure. Please note that Ca V ␣2␦1 was not tagged. D, bar graph summarizing the results shown in A and B with the values for the cell surface density in light gray bars and the total protein density in dark gray bars. Each experimental condition was quantified in triplicate. The fluorescence intensity is shown in arbitrary units. The statistical analysis was performed by comparing the ⌬MFI values measured in all experimental groups versus ⌬MFI values measured for Ca V 1.2-HA alone in intact versus permeabilized cells respectively. **, p Ͻ 0.01; ***, p Ͻ 0.001.
tagged, confirmed the specificity of the FITC antibody in these series of experiments (data not shown). Given that the HA epitope is located in the extracellular portion of the protein, the fluorescence intensity for FITC obtained in the presence of intact cells reflects the cell surface density of Ca V 1.2. Fluorescence for FITC was measured after cell permeabilization to confirm the accessibility of the HA epitope. The fluorescence histograms are reported in Fig. 2, A and B, and the averaged mean fluorescence intensities are shown in Fig. 2D. Ca V ␣2␦ alone improved total protein expression of Ca V 1.2 without any significant change in the cell surface density of Ca V 1.2. This contrasts with the impact of Cav␤ (Fig. 2D) that improves both cell surface and total protein density of Ca V 1.2 (20). In particular, Ca V ␤3 increased by Ϸ200% the cell surface density of Ca V 1.2, whereas Ca V ␣2␦ barely stimulated cell surface density by 2 Ϯ 3% (n ϭ 3). Co-expression with both Ca V ␤3 and Ca V ␣2␦ did not increase cell surface detection of Ca V 1.2, although total protein stability was improved (Fig. 2, A-D). The former results contrast with confocal imaging data showing that co-expression with both Ca V ␣2␦ and Ca V ␤1b is required to achieve the maximal cell surface staining for Ca V 2.2 (39). We had previously reported similar results with an HA-tagged Ca V 1.2 construct bearing its HA epitope in domain I (19).
The flow cytometry data were validated by cycloheximide chase analysis (Fig. 3, A-C). Ca V ␤3 increased the protein expression of Ca V 1.2 in total lysates, whereas Ca V ␣2␦ alone did not significantly alter protein density of Ca V 1.2. As seen in flow cytometry assays (Fig. 2D), total protein expression of Ca V 1.2 was also increased in the combined presence of Ca V ␤3 and Ca V ␣2␦ auxiliary subunits (Fig. 3, D and E), as also reported by others (20).  The protein density of Ca V 1.2 in total lysates was expressed relative to GAPDH and normalized to the protein density measured at time 0. As seen, Ca V ␤3 alone was more effective than Ca V ␣2␦1 in preventing the degradation of Ca V 1.2, but co-expressing Ca V ␤3 with Ca V ␣2␦1 further stabilized the protein density of Ca V 1.2. The time courses of the protein degradation for the following conditions, Ca V 1.2 alone and Ca V 1.2/Ca V ␣2␦1, were indistinguishable from one another and from the ⌬MFI for FITC in the previous figure suggested that Ca V 1.2 (total density) was more stable in the presence of Ca V ␣2␦1. This difference might arise from the higher sensitivity of the fluorescence assays. Molecular mass of Ca V 1.2 is ϳ250 kDa; Ca V ␣2␦1 is 175 kDa; Ca V ␤3 is 60 kDa; and GAPDH is 40 kDa. E, protein density was estimated with ImageJ (rsbweb.nih.gov). The time course of degradation was measured in three series of experiments. Each symbol represents the mean Ϯ S.E. of the normalized protein density of Ca V 1.2.
construct was expressed alone in HEKT and in stable Ca V ␤3 cells in the presence or absence of Ca V 1.2. Two-color flow cytometry experiments were carried out in intact and permeabilized cells to respectively assess cell surface expression and total protein expression, the latter including cell surface and intracellular proteins. In two-dimensional plots, the fluorescence intensity obtained for mCherry was plotted against the fluorescence intensity for FITC. As seen in intact nonpermeabilized cells, the fluorescence intensity was strong for both mCherry and FITC indicating that Ca V ␣2␦ is clearly present at the cell membrane (Fig. 4, A and C). Co-expression with Ca V ␤3 alone did not significantly alter either surface or total expression, whereas co-expression with Ca V 1.2 only slightly improved cell surface and total protein expression of Ca V ␣2␦. However, co-expression with both Ca V 1.2 and Ca V ␤3 nearly doubled the total protein expression of Ca V ␣2␦ but not its cell surface density. It is unlikely that the latter result was due to an unfavorable change in the conformation of the Ca V ␣2␦-HA construct because Ca V 1.2 whole-cell currents were clearly up-regulated by Ca V ␣2␦-HA in patch clamp experiments (see Fig. 1). The relative increase in total protein expression of Ca V ␣2␦ was sim-ilar whether it was inferred from the constitutive mCherry fluorescence of mCherry-Ca V ␣2␦-HA (data not shown) or from the FITC fluorescence in permeabilized cells (Fig. 4D). These results suggest that Ca V ␣2␦ interacts intracellularly with Ca V 1.2 and that the stability of the protein complex is improved by Ca V ␤3, probably in part driven by the nanomolar interaction between Ca V 1.2 and Ca V ␤3 (41). Nonetheless, because the increase in total protein density did not translate into a surge in the cell surface density of Ca V ␣2␦, it also suggests that most of the Ca V 1.2 protein complex remains in intracellular compartments.
Channel Modulation Depends upon the Cell Surface Expression of Ca V ␣2␦-The correlation between the protein expression of Ca V ␣2␦ and the modulation of Ca V 1.2 channels was quantified by co-expressing different DNA ratios of Ca V ␣2␦ and Ca V 1.2. The ⌬MFI values for FITC measured in intact and permeabilized cells increased steeply in the range from 1:20 to a 1:2 DNA ratio. Both the cell surface and the total protein densities followed a similar pattern (Fig. 5). Under our experimental conditions, the 1:1 DNA ratio yielded an additional 20% increase in the protein density, but raising further the relative amount of DNA coding for Ca V ␣2␦ was found to impair trans- Representative two-dimensional plots of mCherry versus FITC fluorescence are shown for each condition as stated (left panels). The vertical line indicates the median fluorescence intensity for FITC to facilitate the visual comparison between the different experimental groups. As seen, the cellular autofluorescence levels increased after permeabilization, which prevents comparison of the absolute fluorescence values between intact nonpermeabilized and permeabilized cells. B, predicted secondary structure of the pmCherry-Ca V ␣2␦1-HA construct used in the figure. Please note that Ca V 1.2 was not tagged either in the N or C termini. C, bar graph shows the ⌬MFI measured in the presence of FITC in intact cells for each experimental condition. Experiments were conducted in triplicate, and each bar is the mean Ϯ S.E. of ⌬MFI in arbitrary units. Under these conditions, ⌬MFI measured for FITC reflects the relative cell surface protein expression of Ca V ␣2␦1. NP, nonpermeabilized; P, permeabilized cells, D, bar graph shows the ⌬MFI measured for FITC in permeabilized cells for each experimental condition. Experiments were conducted in triplicate, and each bar is the mean Ϯ S.E. of ⌬MFI in arbitrary units. Under these conditions, ⌬MFI measured for FITC reflects the total protein expression of Ca V ␣2␦1. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001. fection efficiency (data not shown). Patch clamp experiments established that increasing the cell surface expression of Ca V ␣2␦ improves peak current density and activation gating of Ca V 1.2 (Fig. 6). Peak current density increased as a function of the cell surface density of Ca V ␣2␦ within the range of our experimental conditions (Fig. 7). This suggests that increasing the cell surface density of Ca V ␣2␦ improves the macroscopic activation gating of Ca V 1.2 by promoting the channel activation at more negative potentials.
Cell Surface Density Is Impaired in Ca V ␣2␦ Genetic Variants-To gain further insight into the molecular mechanism underlying channel modulation by Ca V ␣2␦, we turned to mutations of Ca V ␣2␦ associated with cardiac arrhythmias. Burashnikov et al. (29) discovered 23 rare missense variants in three genes encoding subunits forming the Ca V 1.2 L-type calcium channel in 205 patients diagnosed with "J-wave syndromes." Four genetic variants were identified in the CACNA2D1 gene encoding Ca V ␣2␦1. The mutation S709N was found in two unrelated patients. Two mutations (D550Y and Q917H) were identified in the same individual (29), but their expression profile has yet to been fully characterized (32). Ca V ␣2␦ missense mutants D550Y, S709N, Q917H, S956T, the double D550Y/Q917H (29), as well as Ca V ␣2␦ S755T mutant associated with SQTS6 (31) were expressed alone or in combination with Ca V 1.2 and Ca V ␤3, the same subunit composition we used for functional characterization in patch clamp experiments. These six Ca V ␣2␦ mutations were co-expressed with Ca V 1.2 WT (no HA) in the maximum 1:1 ratio in stable Ca V ␤3 cells. The trans-fection efficiency (as assessed by the number of fluorescent cells) was found to be not significantly different between the HA-tagged Ca V ␣2␦ WT and the HA-tagged Ca V ␣2␦ mutant constructs. Overall, the total protein density of all mutants estimated from permeabilized cells or from the constitutive mCherry fluorescence (data not shown) significantly increased in the combined presence of Ca V 1.2 and Ca V ␤3. As shown in Figs. 8 and 9, the ⌬MFI for FITC of Ca V ␣2␦ S755T, Q917H, and S956T mutants in intact cells under these conditions was similar to the ⌬MFI measured for Ca V ␣2␦-HA WT suggesting that the cell surface density of Ca V ␣2␦ was not affected by these single mutations. In contrast, missense mutations D550Y and S709N and more significantly the double mutant D550Y/ Q917H impaired the cell surface targeting of Ca V ␣2␦-HA with Ϸ30% reduction for the former mutants and Ϸ60% decrease for the latter. Doubling the amount of cDNA used for transfection did not improve cell surface expression because the transfection efficiency decreased with larger DNA concentrations. The surface density of D550Y/Q917H proteins remained on average 60% lower than the one measured for the Ca V ␣2␦-HA WT protein at each DNA ratio tested from 1:20 (data not shown) to 1:1. Flow cytometry assays carried out 36 h after transfection yielded similar results suggesting that a 24-h culture time is sufficient to observe the optimal protein expression (data not shown). The fluorescence intensities for FITC in permeabilized cells and for the constitutive mCherry were only decreased by Ϸ30% when compared with the signals measured for Ca V ␣2␦-HA WT. These data indicate the following: (a) HA tag remained accessible in the double mutant; (b) the cell and the total protein density were both affected by the D550Y/ Q917H double mutation albeit to a different extent. Altogether, these data suggest that the double mutation impairs the trafficking of Ca V ␣2␦ to a greater extent than protein stability.
Channel Modulation Is Altered in the Presence of Ca V ␣2␦ Genetic Variants-The functional impact of the missense Ca V ␣2␦ mutations was characterized by electrophysiology after co-expression of Ca V 1.2 and Ca V ␣2␦ in stable Ca V ␤3 cells.
In the presence of a 1:1 ratio, the five single point mutations, including D550Y and S709N, increased Ca V 1.2 peak current densities by 10 -13-fold with a negative shift in the voltage-dependent gating activation (⌬G act Ϸ Ϫ1 kcal mol Ϫ1 ) in a fashion reminiscent of Ca V ␣2␦ WT (Table 1). Hence, Ca V ␣2␦ D550Y and S709N mutations boosted the peak current densities despite a 30% decrease in the cell surface expression of Ca V ␣2␦. These results are in agreement with Fig. 7 showing that an Ϸ30% decrease in the cell surface density of Ca V ␣2␦ is not sufficient to significantly prevent the up-regulation of macroscopic Ca V 1.2 currents. In contrast, co-expression of Ca V ␣2␦-HA D550Y/Q917H produced currents that were 35% lower than Ca V ␣2␦-HA WT with an average of Ϫ40 Ϯ 10 pA/pF (n ϭ 10) compared with Ϫ67 Ϯ 3 pA/pF (n ϭ 163) ( Fig  10). The experimental variation, however, limits the statistical significance to p Ͻ 0.5. Nonetheless, there seems to be a trend toward a decreased function that was also reported by the authors of the original paper (29). We hypothesized that the decrease in the cell surface density of the Ca V ␣2␦ D550Y/ Q917H mutant could become more significant when expressing Ca V ␣2␦/Ca V 1.2 in a 1:20 ratio. Patch clamp experiments  5 g); Ab, ratio 1:4 (1 g); Ac, ratio 1:2.7 (1.5 g); Ad, ratio 1:2 (2 g); and Ae, ratio 1:1 (4 g). Typical whole-cell current traces were recorded in a 2 mM Ca 2ϩ solution from a holding potential of Ϫ100 mV. The pmCherry-Ca V ␣2␦1-HA construct used for the patch clamp experiments was identical to the constructs studied in the flow cytometry assays. Time scale is 100 ms throughout. Unless specified otherwise, the current density scale is 10 pA/pF. B, peak current densities increased from Ϫ5 Ϯ 1 pA/pF (n ϭ 26) (no insert in the pmCherry vector) to Ϫ67 Ϯ 3 pA/pF (n ϭ 163) in the presence of 4 g of cDNA (ratio 1:1) coding for pmCherry-Ca V ␣2␦1-HA WT. The number in parentheses indicates the number of independent patch clamp recordings. C, histogram reporting the distribution of the individual ⌬G act values (kilocalories/mol) for each concentration of cDNA coding for Ca V ␣2␦1. As seen, the ⌬G act are significantly shifted to the left even when Ca V 1.2 is co-expressed with Ca V ␣2␦1 in a 1:1 ratio. D, inactivation kinetics of the Ca V 1.2 currents expressed with Ca V ␣2␦1 in a 1:8 ratio (gray hatched bars) were in general 20% slower (p Ͻ 0.05) than the inactivation kinetics measured after co-expression of Ca V ␣2␦1 in a 1:1 ratio (white striated red bars) (p Ͻ 0.05). FIGURE 7. Correlation between the cell surface density of Ca V ␣2␦1-HA WT and the changes in ⌬G act and peak current density of Ca V 1.2 currents. Peak current density was normalized to the mean value measured with 4 g of cDNA for Ca V ␣2␦1-HA (ratio 1:1); ⌬⌬G act was calculated relative to the value of ⌬G act ϭ 0.48 Ϯ 0.09 kcal mol Ϫ1 for currents measured in the absence of Ca V ␣2␦1; the cell surface density was computed using the normalized ⌬MFI measured for FITC in intact nonpermeabilized cells. Despite the large experimental variation, there is a positive correlation between the cell surface density of Ca V ␣2␦1 and the two other parameters (peak current density and ⌬⌬G act of macroscopic currents). The variation of ⌬⌬G act with the surface density of Ca V ␣2␦1 suggests that Ca V ␣2␦1 influences the fraction of ion channels active in a given gating mode. The correlation could be described by a large number of equations, but it is shown as a linear regression to facilitate visualization.
were thus carried out with cDNA ratios of 1:1, 1:8, and 1:20 Ca V ␣2␦/Ca V 1.2 (0.2:4 g of DNA) (Fig 11). As shown, the behavior of the double mutant departed more significantly from the wild-type protein at lower cDNA concentrations. It is interesting to note that the decrease in peak current density was accompanied by a rightward shift in the activation potentials. Altogether, our results suggest that some Ca V ␣2␦ arrhythmo-genic mutations may decrease Ca V 1.2 currents in cardiomyocytes as a result of their decreased cell surface density.  as well as in native mouse cardiomyocytes (21). Whether this modulation is conveyed through an increase in the cell surface density of Ca V 1.2 remains debated (19,20,42) in part because the macroscopic peak current density is the product of three parameters as follows: the number of channels in the plasma membrane, the open channel probability, and the single-channel conductance. We have opted to investigate changes in the channel surface density using a sensitive and high throughput flow cytometry fluorescence-based assay. In this assay, the cell samples go through a gating process that excludes dead cells, debris, and aggregates such that the fluorescence intensity reflects protein expression in intact cells of similar morphological properties. This approach required the insertion of a 9-residue HA epitope in the extracellular face of the Ca V 1.2 pore-forming subunit. Such manipulation may interfere with protein expression and/or interaction with other subunits as shown very elegantly in Ref. 39. Indeed, we have tested many constructs before identifying one that preserves the up-regulation of Ca V 1.2 currents by Ca V ␣2␦. As Recombinant Ca V ␣2␦ Is Strongly Expressed at the Membrane-The trafficking of Ca V ␣2␦ was studied using a similar fluorescence-based assay, but surface and total protein density were All ⌬MFI values were normalized as compared with the average ⌬MFI measured with Ca V ␣2␦1-HA WT expressed alone in HEKT cells. ⌬MFI for FITC measured in permeabilized cells reflects the total protein expression of Ca V ␣2␦1-HA constructs. As seen, the total protein density decreased by 20 Ϯ 1, 21 Ϯ 2, and 24 Ϯ 11% for Ca V ␣2␦1-HA D550Y, S709N, and D550Y/Q917H, respectively (p Ͻ 0.05), when compared with the wild-type construct. This small decrease in total protein density of these mutants could account in part for their decreased cell surface density. This decrease was also observed for the constitutive fluorescence of mCherry (data not shown). For all mutants, please note that the total protein density of all Ca V ␣2␦1-HA constructs nearly doubled when co-expressed with Ca V 1.2 in stable Ca V ␤3 cells. *, p Ͻ 0.05; ***, p Ͻ 0.001.

TABLE 1 Biophysical properties of Ca V 1.2/ Ca V ␤3 channels with and without Ca V ␣2␦1
Ca V 1.2 WT was expressed in stable Ca V ␤3 cells with pmCherry-no insert or pmCherry-Ca V ␣2␦ construct under typical conditions with a 1:1 DNA ratio (4 g: 4 g). Biophysical parameters were measured in the presence of 2 mM Ca 2ϩ as described elsewhere (19). Activation properties (E 0.5, act and ⌬G act ) were estimated from the mean I-V relationships and fitted to a Boltzmann equation. The data are shown with the mean Ϯ S.E. of the individual experiments, and the number of experiments is in parentheses. ND means not determined as the signal-to-noise ratio was too small in the absence of Ca V ␣2␦1. Ϫ10.9 Ϯ 0.7 (10)

Ca V 1.2 WT in Ca
Ϫ0.9 Ϯ 0.1 (10) Ϫ61 Ϯ 9 (10) 0.33 Ϯ 0.01 (10) ϩ Ca V ␣2␦1-HA S709N (1:1 ratio) Ϫ11.3 Ϯ 0.6 (10) Ϫ0.9 Ϯ 0.1 (10) Ϫ49 Ϯ 10 ( Ϫ10.8 Ϯ 0.6 (10) Ϫ0.9 Ϯ 0.1 (10) Ϫ84 Ϯ 9 (10) 0.33 Ϯ 0.02 (10) ϩ Ca V ␣2␦1-HA D550Y/ Q917H (1:1 ratio) Ϫ9 Ϯ 2 (10) Ϫ0.8 Ϯ 0.2 (10) Ϫ40 Ϯ 10 (10) 0.35 Ϯ 0.03 (10) tracked by the external HA epitope and the intracellular mCherry tag, respectively. The constitutive mCherry fluorescence was used as an index of Ca V ␣2␦ expression under all conditions for the wild-type protein as well as the Ca V ␣2␦ mutants (see below). Patch clamp experiments carried out with the untagged version of Ca V 1.2 but using the HA-tagged mCherry Ca V ␣2␦ constructs (WT and mutants) confirmed the latter construct was functional. It is essential to stress that fluorescence cell sorting experiments (protein expression) and whole-cell recordings (channel function) were carried out with cells grown under the same conditions. These data validated that the double modification, insertion of the HA epitope and the mCherry protein, did not prevent functional modulation of the L-type currents by Ca V ␣2␦. Furthermore, the accessibility of the HA epitope was validated in each flow cytometry assay by comparing the relative fluorescence intensities obtained with the FITC-conjugated antibody in permeabilized cells with the constitutive mCherry in intact and permeabilized cells. Ca V ␣2␦ was shown to reach the cell surface when expressed alone (our data in HEKT cells and see Ref. 42). The strong fluorescence intensity suggests that the cell surface density of Ca V ␣2␦ is larger than the cell surface density of Ca V 1.2 expressed under similar conditions. Similar observations were made in mouse cardiomyocytes showing that endogenous Ca V ␣2␦ is significantly expressed at the plasma membrane ( Fig  12). Co-expression with Ca V ␤3 did not appreciably alter the cell surface density of Ca V ␣2␦ in contrast to what we and others have reported for the Ca V ␣1 subunit of Ca V 1.2 (19). Nonetheless, our study highlighted some reciprocal modulation within the proteins forming the L-type Ca V 1.2 channel. Indeed, the total protein expression of Ca V ␣2␦ significantly improved in the combined presence of Ca V 1.2 and Ca V ␤3. The significant increase of total protein expression in the combined presence of Ca V 1.2 and Ca V ␤3 measured from the relative fluorescence for FITC in permeabilized cells did not result from an improved accessibility of the HA epitope because the constitutive mCherry fluorescence as well as the total protein density measured in Western blots were similarly augmented. Furthermore, our results show that the modulation of L-type currents is a quasi-linear function of the cell surface density of Ca V ␣2␦. It suggests that the interaction between Ca V 1.2 and Ca V ␣2␦ is conveyed through one or multiple low affinity binding site(s).
Cardiac Arrhythmias and Ca V ␣2␦-In the context where the numbers of channelopathy susceptibility genes and mutations identified are increasing rapidly, there is renewed interest in characterizing the molecular mechanisms underlying modulation of L-type Ca V 1.2 channels by its auxiliary subunits, in particular Ca V ␣2␦. In return, structure-function insights gleaned from these disease susceptibility genes have significantly advanced our understanding of the pathophysiology of these syndromes and have paved the way to the development of new treatment strategies. Over the last few years, single missense mutations and/or genetic variants of Ca V ␣2␦ have been identified in patients experiencing cardiac arrhythmias associated with repolarizing QT interval (SQT) shorter than normal. Short QT arrhythmias are generally associated with gain-of-function outward currents (potassium currents) or loss-of-function of inward (sodium or calcium) currents. At the time of their identification, the missense mutations in Ca V ␣2␦ have been labeled loss-of-function mutations and were proposed to alter L-type Ca V 1.2 peak current density through a change in the membrane expression of Ca V 1.2 (29). Having established a reliable experimental model to define the surface expression and the function of the different components of the L-type Ca V 1.2 channel, we explored the impact of Ca V ␣2␦ mutations associated with sudden cardiac death. Recombinant expression in HEKT cells of Ca V ␣2␦ single mutants S755T, Q917H, and S956T associated with shorter QT intervals did not cause any significant change in the cell surface expression of Ca V ␣2␦ or in the function of Ca V 1.2 currents when expressed using a 1:1 cDNA ratio. This is true also for the mutation Ca V ␣2␦ S755T that was reported by others to prevent the up-regulation of Ca V 1.2 currents by Ca V ␣2␦ in HEKT cells (31). In contrast, we have observed a, Ϸ30% decrease in the cell surface density of Ca V ␣2␦ D550Y and S709N single mutants accompanied with an Ϸ15% in the total protein density that remained unchanged up to 36 h after transfection. Despite causing a reduction in the cell surface expression of Ca V ␣2␦, the single mutants D550Y and S709N did not significantly impair the modulation of Ca V 1.2 currents when co-expressed in a 1:1 cDNA ratio. Combining two mutations in Ca V ␣2␦ with D550Y/Q917H significantly reduced the cell surface density of Ca V ␣2␦ (60%) under the same conditions. Although both the cell surface and the total protein density of the double mutant were reduced, the decrease in the cell surface Ab, Ca V ␣2␦-HA D550Y/Q917H; Ac, average peak current densities increased from Ϫ5 Ϯ 1 pA/pF (n ϭ 26) (no insert in the pmCherry vector) to Ϫ7 Ϯ 1 pA/pF (n ϭ 9) for mCherry-Ca V ␣2␦1-HA D550Y/Q917H, and Ϫ17 Ϯ 2 pA/pF (n ϭ 4) for WT (p Ͻ 10 Ϫ6 ). Co-expression with Ca V ␣2␦ D550Y/Q917H significantly increased peak current densities of Ca V 1.2 as compared with the currents obtained in the absence of Ca V ␣2␦ but were significantly different from Ca V ␣2␦1 WT (p Ͻ 0.05). The ⌬G act (in kcal mol Ϫ1 ) values were Ϫ0.02 Ϯ 0.01 for D550Y/Q917H and Ϫ0.30 Ϯ 0.05 for WT. B, DNA ratio for Ca V ␣2␦/Ca V 1.2 of 1:8 with 0.5 g of DNA for Ca V ␣2␦-HA. Ba, Ca V ␣2␦-HA WT; Bb, Ca V ␣2␦-HA D550Y/Q917H; Bc, average peak current densities were Ϫ18 Ϯ 3 pA/pF (n ϭ 12) for mCherry-Ca V ␣2␦1-HA D550Y/Q917H, and Ϫ36 Ϯ 5 pA/pF (n ϭ 12) for WT. Co-expression with Ca V ␣2␦ D550Y/Q917H significantly increased peak current densities of Ca V 1.2 as compared with the currents obtained in the absence of Ca V ␣2␦ but were significantly different from Ca V ␣2␦1 WT (p Ͻ 0.05). The ⌬G act (in kilocalories mol Ϫ1 ) values were Ϫ0.29 Ϯ 0.03 for D550Y/Q917H and Ϫ0.57 Ϯ 0.09 for WT. C, DNA ratio for Ca V ␣2␦/Ca V 1.2 was 1:1 with 4 g of DNA for Ca V ␣2␦. Ca, Ca V ␣2␦1WT; Cb, Ca V ␣2␦1 D550Y/Q917H; Cc, average peak current densities are shown under these conditions. QY/QH should read DY/QH for D550Y/Q917H. Numerical values are shown in Table 1. Co-expression with Ca V ␣2␦ D550Y/Q917H significantly increased peak current densities of Ca V 1.2 as compared with the currents obtained in the absence of Ca V ␣2␦ and were significantly different from Ca V ␣2␦1 WT (p Ͼ 0.1).
expression was more important than the reduction in the total protein density. Altogether, these data suggest that the trafficking of Ca V ␣2␦ was significantly disrupted by the double mutation. The decrease in the expression of Ca V ␣2␦ caused a significant 40% reduction in the peak current density of Ca V 1.2 when co-expressed with Ca V ␣2␦ WT in stable Ca V ␤3 cells. Hence, the combination of two variants that are relatively silent polymorphisms on their own could significantly impair the trafficking of Ca V ␣2␦ and consequently reduce L-type Ca V 1.2 currents.
Conclusion-Current genetic studies are placing the homeostasis of Ca 2ϩ as a central modulator of cardiac repolarization with genes such as CACNA1C as well as ATP2A2, PLN, PRKCA, SRL, and SLC8A1 (2). Genetic studies have reported a decreased expression of Ca V ␣2␦ transcripts in patients suffering from atrial (43) and ventricular fibrillation (44). Our current results confirm that mutations in CACNA2D1 could be a contributing factor in cardiac sudden death associated with a short QT interval. It is important to note that the strongest reduction in the cell surface density of Ca V ␣2␦ was observed by combining two genetic variants that had little impact when tested individually. Hence, the role of polymorphisms in CACNA2D1 is not to be neglected. Missense and/or truncation mutations of Ca V ␣2␦ could severely impair Ca V 1.2 currents providing that the said mutation significantly decreases the cell surface protein expression of Ca V ␣2␦.