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CARDIOVASCULAR

Design of Mutant β₂ Subunits as Decoy Molecules to Reduce the Expression of Functional Ca²⁺ Channels in Cardiac Cells

Departments of Internal Medicine (S.T., T.G., J.D.M.) and Pharmacology and Toxicology (S.S., S.W.R., J.R.S., N.J.R.), College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas

	Abstract

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Calcium influx through long-lasting ("L-type") Ca²⁺ channels (Ca_V) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca²⁺ overload, and interventions that reduce expression of the pore-forming ₁ subunit may alleviate cytosolic Ca²⁺ excess. As a molecular approach to disrupt the assembly of Ca_V1.2 (_1C) channels at the cell membrane, we targeted the Ca²⁺ channel β₂ subunit, an intracellular chaperone that interacts with _1C via its β interaction domain (BID) to promote Ca_V1.2 channel expression. Recombinant adenovirus expressing either the full β₂ subunit (Full-β₂) or truncated β₂ subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-β₂ at the surface membrane was associated with increased Ca²⁺ current mainly attributed to Ca_V1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the _1C protein of Ca_V1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca²⁺ current. C-BID and BID also attenuated Ca²⁺ current associated with another L-type Ca²⁺ channel, Ca_V1.3, but they did not reduce transient Ca²⁺ currents attributed to Ca_V3 channels. These results suggest that β₂ subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca²⁺ channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca²⁺ overload.

Although physiologically essential to normal cardiac function, Ca_V1.2 channels also have been implicated in pathological processes involving Ca²⁺ overload, including cardiac ischemia and myocardial stunning. In these conditions, an elevated cytosolic Ca²⁺ ([Ca²⁺]_cyt) in the myocardial cells is associated with a loss of myofilament sensitivity to Ca²⁺ (Bolli and Marban, 1999). Moreover, Ca²⁺ overload also plays an important role in cardiac apoptosis and necrosis (Aon et al., 2003). Even under conditions in which [Ca²⁺]_cyt is within a normal range, it may be desirable to modestly depress [Ca²⁺]_cyt as a therapeutic intervention to slow atrioventricular conduction in atrial fibrillation or to modestly depress contractility in hypertrophic cardiomyopathy. Currently, calcium channel blockers represent the drug therapies of choice for these conditions, but several features of their properties, including their short duration of action and vascular dilator effects, limit their usefulness.

In this respect, a gene therapy approach designed to reduce the availability of β₂ subunits required for the expression of Ca_V1.2 channels may provide for the highly targeted and long-term relief of calcium overload. Accordingly, we engineered recombinant adenoviral constructs expressing truncated β₂ to provide proof of concept that mutated β₂ could potentially act as a dominant-negative construct to reduce Ca_V1.2 channels at the surface membrane. In the present study, we expressed the recombinant adenovirus constructs in HL-1 cells, a cell line derived from AT-1 mouse atrial tumor lineage that displays many normal markers of cardiac biology. HL-1 cells spontaneously depolarize and express ion channels required for generating action potentials (White et al., 2004), and they retain the signaling pathways to respond appropriately to inotropic and chronotropic agonists (Claycomb et al., 1998). The activity of the truncated β₂ constructs was compared with the effect of full-length β₂ (Full-β₂), which we have shown enhances the expression of functional Ca_V1.2 channels in feline cardiac myocytes (Chen et al., 2005). The goal was to retain the ability of the β₂ decoys to bind to _1C but to eliminate the amino and/or carboxyl termini of β₂ that may be required for the expression of functional _1C subunits in the plasma membrane, thereby "trapping" Ca_V1.2 channels inside the cell. We also evaluated the effect of the β₂ subunit mutants on Ca²⁺ current attributed to two other types of Ca²⁺ channels important in pacemaker activity or contractile function. The Ca_V1.3 channels (_1D) mediate L-type current at mid-voltages, and they are approximately 10-fold less sensitive to 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (nifedipine)-induced block than Ca_V1.2 channels at negative membrane potentials (Koschak et al., 2001). HL-1 cells also express Ca_V3 channels that activate at much lower voltages than L-type channels, and they mediate transient ("T-type") current associated with normal pacemaker activity in cardiac cells (Xia et al., 2004; Zhang et al., 2005).

	Materials and Methods

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Culture of HL-1 Cells. HL-1 cells (kindly provided by Dr. W. C. Claycomb, Louisiana State University, Baton Rouge, LA) were grown and maintained in Claycomb medium (SAFC Biosciences, Lenexa, KS) supplemented with penicillin-streptomycin (1x) and 2 mM L-glutamine (Invitrogen, Carlsbad, CA). After trypsinization, dissociated cells were either plated in standard 60-mm tissue culture dishes (0.8–1.0 x 10⁶ cells/ml) for mRNA and/or protein expression assays or in glass culture dishes (MatTek, Ashland, MA) for electrophysiology (60–80 x 10³ cells/ml). Cells were then infected for 24 to 48 h with one of four different GFP-fused β₂ subunit constructs, including Full-β₂, or truncated β₂ lacking either the N terminus (C-BID), the C terminus (N-BID), or both termini (BID only) or with the control GFP construct. The optimal dose of crude viral stock (multiplicity of infection, 50–100) needed to optimize protein expression without visible cell death was determined by fluorescent imaging and immunoblot techniques. Experimental conditions were developed that produced >95% infection of HL-1 cells for each of the constructs studied, as described previously (Fan et al., 2003; Telemaque-Potts et al., 2003; Chen et al., 2005). All studies on HL-1 cells were performed between 24 and 48 h after transfection.

Detection of Ca_L Channel Subunit Gene Expression in HL-1 Cells. To identify the endogenous β subunits expressed in HL-1 cells, total RNA was isolated from untreated HL-1 cells using a TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). Primers for PCR reactions were designed to detect Ca_V1.2 (_1C) and different β subunit isoforms (β₁, β_2a, β_2b, β₃, and β₄). Sense and antisense primers were as follows: _1C:5'-CCGCCCACTACCAAGATCAAC-3', 5'-TCGTGTCATTGACAATGCGG-3', covering cDNA bases 2536 to 2791 (256 bp; GenBank accession number NM_012517 [GenBank] .1 [rat]); β₁:5'-TCCAGAAGAGCGGCATGTCCC-3', 5'-AGGACGTACTCCCGTCTGAC-3', covering cDNA bases 5 to 140 (136 bp; GenBank accession number NM_031173 [GenBank] .2 [mouse]); β_2a: 5'-CGAGTACGGGTGTCCTATG, 5'-CGTCCTATCCACCAGTCAT-3', covering cDNA bases 31 to 320 (290 bp; GenBank accession number NM_053851 [GenBank] .1 [rat]); β_2b: 5'-AGGCAGTTGGTGTCTTCTC-3', 5'-CGTCCTATCCACCAGTCAT-3', covering cDNA bases 10 to 323 (314 bp; GenBank accession number AF423193 [GenBank] [rat]); β₃:5'-CATCCCTGGACTTCAGAAC-3', 5'-TGGTAGGCATCTGCATAGTC-3', covering cDNA bases 1125 to 1301 (177 bp; GenBank accession number NM_007581 [GenBank] .2 [mouse]); and β₄:5'-CTCACCATATCCCACAGCAA-3', 5'-TCCGGGTAATCTTCTTCCACCA-3', covering cDNA bases 1266 to 1478 (213 bp; GenBank accession number NM_001037099.1 [mouse]).

Reactions contained 2 µl of cDNA, 10 pmol of each primer, 0.3 mM dNTPs, 1.0 mM MgSO₄, and 0.5 units of Pfx DNA polymerase (Invitrogen) mixed with 1x enhancer buffer in a total volume of 25 µl. Amplification steps included initial denaturation at 94°C for 3 min, followed by 30 cycles (denaturation for 30 s at 94°C; annealing 30 s at 53°C; extension 30 s at 68°C). A final step for product extension was performed at 68°C for 5 min. The PCR products (5 µl) were separated using a 1.2% agarose gel stained with ethidium bromide for visualization. All oligonucleotide sequences were directed toward the mouse/rat sequences of the Ca²⁺ channel subunits.

Generation of β₂ Subunit Constructs. A plasmid encoding the rat heart/brain β_2a subunit cDNA, a generous gift from Dr. E. Perez-Reyes (Perez-Reyes et al., 1992), was used as a template for generating three PCR-based mutations of the β_2a subunit. In brief, cDNA encoding the N-BID fragment (bp 376-1177), BID fragment (bp 867–1177), and C-BID fragment (bp 867-2427) were amplified, digested, and directionally cloned into pEGFP-C1 vector (Clontech, Mountain View, CA), an expression vector driven by a cytomegalovirus (CMV) promoter. All β₂ subunit constructs were cloned in frame with an enhanced GFP reporter gene located at the amino-terminal end of the construct. A schematic representation of the various β₂ subunit adenoviral constructs is illustrated in Fig. 1.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Schematic representation of rat cDNA constructs expressing Full-β2 and truncated β2 subunits N-BID, BID, and C-BID. EGFP, enhanced GFP.

Preparation of Recombinant Adenovirus Construct. Recombinant adenovirus constructs were prepared based on the methods described by He et al. (1998) and made commercially available (AdEASY) by Stratagene (La Jolla, CA). The CMV-GFP-β subunit fragments were gel-purified and subcloned into the pShuttle vector. Then, it was linearized and cotransformed into Escherichia coli BJ5183 cells with pAdEasy-1, the adenoviral backbone. A CMV-GFP lacking any β subunit construct also was subcloned into a pShuttle vector to construct a control virus (Fan et al., 2003). Recombinants were then linearized and transfected into human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) using FuGENE 6 reagent (Roche Molecular Systems, Alameda, CA). Virus-containing medium from HEK 293-infected cells was collected 3 to 6 days after infection, and proper expression of GFP-fused β subunits was confirmed by PCR and immunodetection. High-titer viral stock was generated by several rounds of amplification in HEK 293 cells and by purification by cesium chloride gradient ultracentrifugation (Niranjan et al., 1996; Telemaque-Potts et al., 2003). Final viral stock titer was determined by plaque assay (1–3 x 10⁸ plaque-forming unit/µl).

Immunodetection. Forty-eight hours after infection, cells were washed twice with ice-cold phosphate-buffered saline (PBS), and then they were lysed in Triton lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl₂, 1% Triton X-100, 1% glycerol, 1x Sigma phosphatase inhibitor cocktail, and 1x Sigma protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell lysates were incubated on ice for 15 to 20 min and centrifuged at 4°C for 15 min at 14,000g. The supernatants were then collected. Protein concentration in supernatants was measured by the Bradford method (Bio-Rad, Hercules, CA). Equal protein samples (40 µg of total protein) were loaded on 3 to 8% NuPAGE Tris acetate gels (Invitrogen), and they were run and transferred to a polyvinylidene fluoride membrane. After blocking the membrane (Tris-buffered saline solution containing 10% nonfat dry milk and 0.1% Tween 20, immunoblotting was performed using mouse _1C monoclonal antibody (1:200; NeuroMabs, Davis, CA), mouse β-actin monoclonal antibody (1:5000; Sigma-Aldrich), and mouse GFP monoclonal antibody (1:2000; Invitrogen). Corresponding secondary horseradish peroxidase-conjugated antibodies were used (1:10,000–1:20,000 dilutions; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Additional Western blots were probed with polyclonal antibodies using methods described previously (Pesic et al., 2004). These antibodies were obtained commercially (Alomone Labs, Jerusalem, Israel) and directed against Ca_V1.2 (_1C), Ca_V1.3 (_1D), Ca_V3.1 (_1G), and Ca_V3.2 (_1H). Protein bands were visualized by enhanced chemiluminescence, and the blots were exposed to Hyperfilm (GE Healthcare, Chicago, IL). Band densities were quantified by densitometry (Gel-Doc System; Bio-Rad), analyzed with Bio-Rad image analysis software, and they were normalized to β-actin expression.

Fluorescent Localization of Recombinant Adenovirus Proteins. Infected cells grown on glass-bottomed tissue culture dishes for 24 h after infection were washed, fixed in 4% paraformaldehyde, and kept in the dark at 4°C until fluorescent imaging. The images were acquired using a spinning disk confocal CARV II unit (BD Biosciences, San Jose, CA) on a Zeiss 200M microscope (Carl Zeiss, Thornwood, NY) fitted with a 100x/1.4 numerical aperture objective and a Retiga EXi camera (QImaging, Surrey, BC, Canada).

Whole-Cell Patch Clamp. Functional expression of Ca²⁺ channels was evaluated using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). After 24 h of infection with the different β₂ constructs, culture dishes were mounted on the stage of an inverted microscope (Nikon, Tokyo, Japan), and HL-1 cells expressing GFP were identified by epifluorescence microscopy and selected for electrophysiological recordings. Cells were initially bathed in a solution containing 145 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 5.5 mM dextrose. After formation of a conventional gigaohm seal, the patched membrane was ruptured to obtain the whole-cell configuration. Cells were then bathed in a solution that consisted of 20 mM BaCl₂, 115 mM N-methyl-D-glucamine, 10 mM HEPES, 1 mM MgCl₂, and 5.5 mM dextrose (pH 7.4; 300 mOsm). The pipette solution consisted of 120 mM cesium aspartate, 30 mM cesium chloride, 0.5 mM GTP, 5 mM MgATP, 5 mM creatine phosphate, and 5 mM sodium pyruvate (pH 7.2; 300 mOsm). Data acquisition and pulse generation were performed using pClamp software (Molecular Devices, Sunnyvale, CA) and an Axopatch 200A amplifier interfaced with a Digidata-1320 acquisition system (Molecular Devices). The data were sampled at 2 to 10 kHz, and they were filtered at 1 to 5 kHz. Capacitance and series resistance compensations were used, and leak current was linearly subtracted using a P/4 protocol, with a subtracting holding potential of –100 mV. Currents were normalized to cell size by dividing by cell capacitance (in pA/pF). Data analysis was performed using Clampfit (Molecular Devices) and Origin (OriginLab Corp., Northampton, MA), as described previously (Stimers et al., 2003). Activation curves were fit by the Boltzmann equation: I/I_max = A₂ + (A₁ – A₂)/(1 + exp((V – V_1/2)/s), where V is the command voltage, V_1/2 is the potential of half-maximal activation, s is the slope factor, A₁ is the maximum value of the ratio I/I_max (fixed at 1), and A₂ is the minimum value of the ratio I/I_max. Steady-state inactivation curves also were fit according to the Boltzmann function I/I_max = A₂ + (A₁ – A₂)/(1 + exp((V – V_1/2)/s), to obtain half-maximal inactivation values.

Chemicals. Unless otherwise stated, all standard chemical reagents were purchased from Sigma-Aldrich.

Statistical Analysis. Data are reported as mean ± S.E.M. Statistical comparisons were performed using one-way analysis of variance followed by post hoc Student-Newman-Keuls test. Values of P less than 0.05 were considered significant.

	Results

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Characterization of Ca²⁺ Channel Subunits in HL-1 Cells. Before introducing β subunit decoys, we determined the native profile of β subunits in HL-1 cells. Transcripts for the Ca_V1.2 (_1C) subunit and for multiple β subunits, including β₁, β₂, β₃, and β₄, were detected (Fig. 2A). Subsequently, we confirmed the presence of nifedipine-sensitive, high-threshold Ca²⁺ channel current (I_Ca) attributed to Ca_V1.2 channels in these cells. Exposure of HL-1 cells to 10 µM nifedipine (n = 4) resulted in a marked inhibition of I_Ca elicited by stepwise 10-mV depolarizing pulses between –40 to +50 mV (Fig. 2B). In these recordings, the I_Ca resistant to block by nifedipine was attributed to the presence of Ca²⁺ channel types other than Ca_V1.2 (Xia et al., 2003; Hansen et al., 2004; Zhang et al., 2005).

View larger version (39K): [in this window] [in a new window]   Fig. 2. A, agarose gel of PCR products corresponding to the 1C subunit of CaV1.2 and native β1, β2a, β2b, β3, and β4 subunits in HL-1 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, representative whole-cell ICa recorded from an HL-1 cells before (left) and after (right) exposure to 10 µM nifedipine for 2 min. Similar results were obtained in three other nifedipine-treated cells.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Immunoblot of total cell lysates from HL-1 cells (40 µg protein/lane) infected with five different adenoviruses expressing GFP alone or GFP-fused β2 subunit constructs. Lane 1, untreated cells; lane 2, cells infected with Full-β2 (106 kDa); lane 3, C-BID (88 kDa); lane 4, N-BID (66 kDa); lane 5, BID (42 kDa); and lane 6, GFP (30 kDa). A, immunodetection using GFP antibody. B, gel blots depicting expression of 1C (240- and 200-kDa bands) and β-actin (42 kDa, internal loading control) using specific antibodies. Data are shown as mean ± S.E.M. of six independent experiments.

Cellular Localization of Various β₂ Subunit Adenoviral Constructs. Cells were examined by fluorescence microscopy 24 h after treatment with adenoviral vectors. Preliminary experiments demonstrated that for adenoviral infection with either the Full-β₂ construct or various mutated constructs, gene expression determined by GFP fluorescence was already present 24 h after viral infection. Maximal in vivo fluorescence was observed by 48 h, which was confirmed by immunoblots. Although the duration of gene expression was not formally investigated in this study, strong fluorescence was maintained for at least 5 days under similar experimental conditions. We postulated that overexpression of Full-β₂ subunit would promote the normal trafficking of Ca_V1.2 channels to the plasma membrane. However, we anticipated that truncated β₂ subunits would retain the ability to bind to the _1C subunit but display abnormal protein trafficking, and thereby not localize Ca_V1.2 channels at the surface membrane. Compared with untreated HL-1 cells (Fig. 4A), confocal images of HL-1 cells infected with GFP revealed a diffuse pattern of fluorescence in the cytosol (Fig. 4B), as reported previously in isolated cardiomyocytes (Telemaque-Potts et al., 2003; Chen et al., 2005). In contrast, overexpression of Full-β₂ produced sarcolemmal localization (Fig. 4C, arrows), whereas C-BID and N-BID fusion proteins seemed primarily localized to intracellular vesicles (Fig. 4, D and E, respectively). Overexpression of BID produced a diffuse reticular pattern suggestive of cytosolic localization (Fig. 4F). Thus, the truncated β₂ subunits showed punctate or diffuse intracellular localization that was distinctly different from the sarcolemmal localization of full-length β₂. We also attempted to localize endogenous _1C subunit by immunostaining, using two different commercially available antibodies against Ca_V1.2 channels, but we were unable to detect a specific signal at the cell membrane (Supplemental Figs. A and B).

View larger version (66K): [in this window] [in a new window]   Fig. 4. Fluorescence microscopic images of HL-1 cells show a different localization pattern for each β2 subunit construct at 24 h after infection. Untreated cells (A) and cells infected with GFP (B), Full-β2 (C), C-BID (D), N-BID (E), and BID (F). Arrows indicate the cell membrane localization of fluorescence. Scale bar, 10 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Effect of adenovirus-mediated overexpression of Full-β2 subunit and different truncated β2 subunit constructs on ICa primarily attributed to CaV1.2 channels in HL-1 cells. A, representative raw current traces (–30 to +20 mV) of whole-cell ICa measured in cells expressing GFP (A), Full-β2 (B), N-BID (C), C-BID (D), or BID (E). Average I-V data were normalized to cell capacitance. Cells were held at –40 mV, and they were given a series of 200-ms pulses from –40 to +50 mV, using progressive 10-mV depolarizing steps. F, I-V relationship shows ICa as a function of membrane potential at which it was elicited in cells expressing GFP (), Full-β2 (), N-BID (), C-BID (), or BID (). Average peak ICa was plotted versus voltage. Data are shown as mean ± S.E.M. , P < 0.05 between Full-β2-infected cells and cells infected with GFP alone (n = 10–12). *, P < 0.05 between BID-infected cells and cells infected with GFP alone (n = 10 each). #, P < 0.05 between C-BID-infected cells and cells infected with GFP alone (n = 7–10).

Effect of Overexpression of C-BID and BID on Voltage-Dependent Activation and Inactivation of I_Ca. Although we hypothesized that C-BID and BID reduced I_Ca in HL-1 cells by retaining Ca_V1.2 channels intracellularly, we investigated the possibility that voltage-dependent gating also was altered. The voltage dependence of activation was evaluated by holding cells at –55 mV and imposing 10-mV depolarizing steps between –50 and +20 mV. Peak current at each voltage was plotted as a ratio of maximal current elicited at +20 mV, and the data were fit by a Boltzmann equation to obtain V_1/2. The resulting activation curves in Fig. 6A revealed no significant difference (n = 4–5) in V_1/2 values between cells overexpressing GFP (–14 ± 1 mV), C-BID (–12 ± 1 mV), or BID (–13 ± 1 mV). Inactivation was assessed by subjecting cells to 1400-ms prepulse voltages (10-mV steps) between –50 and 30 mV from a holding potential of –55 mV. Cells were briefly returned to –55 mV for 20 ms, and then they were pulsed to +20 mV to assess the effect of inactivating prepulses on channel availability. Peak current at each prepulse voltage was plotted as the ratio of maximal current elicited from a holding potential of –55 mV. Figure 6B shows data fit with the Boltzmann function, and it reveals that V_1/2 values were not significantly different (n = 4–5) for HL-1 cells overexpressing GFP (–7 ± 2 mV), C-BID (–3 ± 2 mV), and BID (–5 ± 1 mV).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effect of adenovirus-mediated overexpression of different truncated β2 subunit constructs on voltage-dependent activation and inactivation of Ca2+ channels primarily attributed to CaV1.2. A, activation curve showing normalized currents elicited at various command potentials (–50 to +20 mV) from a holding potential of –55 mV. I/Imax value was calculated at each voltage, and it was plotted against the voltage. The solid lines represent fits with the Boltzmann function. There was no significant difference in V1/2 between cells overexpressing GFP compared with C-BID and BID (n = 4–5). B, voltage-dependent inactivation of Ca2+ channel currents using a double-pulse protocol. Prepulse voltage steps (–50 to 30 mV) from a holding potential of –55 mV were imposed, and channel availability evaluated by a subsequent voltage pulse from –55 to +20 mV. I/Imax value was calculated at each voltage and plotted against the voltage. Data fit with the Boltzmann function revealed similar half-inactivation voltages for GFP-, C-BID-, and BID-overexpressing cells. Data values are expressed as mean ± S.E.M. (n = 4–5).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Calcium current separation using nifedipine to determine the effect of C-BID and BID overexpression on different Ca2+ current components in HL-1 cells. Currents were elicited using 200-ms depolarizing steps (–60 to 50 mV) from a holding potential of –80 mV, in 10-mV increments. The currents were normalized to cell capacitance. The resulting I-V relationships are shown for cells overexpressing GFP, BID, or C-BID before (A, total current) and after (B, nifedipine-resistant current) exposure of cells to 10 µM nifedipine. C, I-V relationship for nifedipine-sensitive Ca2+ current was obtained by subtracting the nifedipine-resistant current from total current. All I-V relationships (A–C) were significantly suppressed by overexpression of BID and C-BID compared with the control construct GFP (n = 4–5 each point).

Based on these findings, we questioned whether C-BID and BID act nonspecifically to suppress both T-type and L-type Ca²⁺ currents. It is noteworthy that the surface localization of L-type channels, including Ca_V1.2 and Ca_V1.3, is thought to rely at least partly on β subunits (Catterall et al., 2005). In contrast, T-type channels are missing the known motifs required for β subunit interaction (Perez-Reyes, 1998, 2003; Catterall et al., 2005). To confirm the expression of both Ca_V1.3 and Ca_V3 channels in the HL-1 cells, and thus a possible contribution to nifedipine-resistant I_Ca, we probed Western blots with ₁ subtype-specific antibodies using mouse brain as a positive control in each of the left lanes (Fig. 8A). In addition to detecting Ca_V1.2, ₁ subunits corresponding to Ca_V1.3, Ca_V3.1 and Ca_V3.2 were evident. Subsequently, we applied a depolarizing pulse between –80 and –10 mV to cells overexpressing GFP, C-BID, or BID to maximally activate low-threshold, T-type (Ca_V3) channels but avoid activation of mid- (Ca_V1.3) and high (Ca_V1.2)-threshold L-type channels (Fig. 8B, top traces). To further isolate T-type current, (1S,2S)-2-[2[[3-(2-benzimidazolylpropyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate (mibefradil) (7 µM), a preferential blocker of T-type channels (Martin et al., 2000; Xia et al., 2003) was applied and peak mibefradil-sensitive current elicited at –10 mV was calculated by digital subtraction (Fig. 8B, bottom traces). This transient current was fully intact after BID and C-BID overexpression, suggesting that the decoy effect of the β₂ subunit mutants does not extend to T-type channels (Fig. 8). Rather, the dominant-negative effect of C-BID and BID on nifedipine-resistant current apparently reflects an action on Ca_V1.3 channels or other Ca²⁺ channel types.

View larger version (27K): [in this window] [in a new window]   Fig. 8. A, Western blot confirming that HL-1 cells express L-type (CaV1.2 and CaV1.3) and T-type (CaV3.1 and CaV3.2) Ca2+ channels. The molecular masses for the proteins of interest were CaV1.2, 190 to 240 kDa; CaV1.3, 250 kDa; CaV3.1, 240 kDa; and CaV3.2, 230 kDa. Br, brain. B, low-threshold current was elicited by progressive 10-mV pulses from –80 to –10 mV in control solution in HL-1 cells overexpressing GFP, C-BID, and BID (top traces). The transient current was reduced by 7 µM mibefradil, a preferential blocker of T-type channels (bottom traces). C, bar diagram shows the peak mibefradil-sensitive Ca2+ current density at –10 mV, which was calculated as the difference between the peak Ca2+ current density at –10 mV before and after the treatment with mibefradil. There was no significant difference in mibefradil-sensitive Ca2+ current among the three groups of cells (n = 4–5).

	Discussion

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In the present study, we also examined the dominant-negative effects of C-BID and BID on additional Ca²⁺ channels coexpressed in HL-1 cells with Ca_V1.2. In our cells, mibefradil-sensitive, T-type current was not attenuated by either β₂ subunit mutant, consistent with reports that Ca_V3 channels do not have binding motifs to permit β subunit interaction (Perez-Reyes, 1998, 2003; Catterall et al., 2005). However, other components of nifedipine-resistant current were suppressed by gene delivery of C-BID and BID. One target probably was the Ca_V1.3 channel that provides L-type current at mid-voltages in HL-1 cells, and it is 10-fold less sensitive to nifedipine than Ca_V1.2 channels. Although precise identification of Ca_V1.3 current is difficult due to the lack of a specific antagonist, members of the Ca_V1 gene family, including Ca_V1.3, share an -interactive domain that enables ₁-β interaction (Catterall et al., 2005). Thus, a shared feature of the Ca_V1 gene family may be susceptibility to dominant-negative β₂ mutants. In this regard, one potential therapeutic benefit of our mutants may include the treatment of supraventricular tachyarrhythmias, because atrial cells seem to densely express Ca_V1.3 channels, which contribute to normal rhythmicity (Zhang et al., 2005). In contrast, Ca_V1.3 channels are only sparsely expressed in ventricular tissues, and Ca_V1.2 channels seem to be the primary pathway for voltage-gated Ca²⁺ influx. Thus, disruption of expression of Ca_V1.2 channels by our β₂ subunit mutants may be beneficial in pathologies involving Ca²⁺ overload as a strategy to depress [Ca²⁺]_cyt and restore normal contractility.

Our findings also provide initial insight into the interactions between the _1C subunit and truncated β₂ subunit decoys that may be required to interrupt the trafficking of functional Ca_L channels to the surface membrane. In contrast to the observation that overexpression of the full-length β₂ subunit at the surface membrane was associated with increased I_Ca, the highly distinctive vesicular patterns for N-BID and C-BID localization and the reticular pattern for BID suggest disruption of proper localization of Ca_L channel subunits. Indeed, each of the truncated β₂ subunits was designed to retain the central BID sequence necessary for _1C-β interaction, but at least one of the two additional protein-interaction domains (SH3 and GK) recently reported to be required for channel assembly was eliminated (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). Thus, the SH3 domain was absent in C-BID, a significant portion of the GK domain was missing in N-BID, and the SH3 domain and a significant portion of the GK domain were missing in BID. In each case, the localization of β₂ subunits was abnormal, supporting previous reports that the β subunits rely on at least three domains in the molecule to confer structural integrity.

Although all three truncated β₂ subunits resulted in altered cellular localization, only C-BID and BID acted as dominant-negative subunits by markedly depressing I_Ca in the HL-1 cells. It seems logical to assume that the overexpression of the two successful β₂ mutants, C-BID and BID, overwhelmed interactions between the endogenous _1C and β subunits to sequester _1C subunit. However, the abnormal vesicular localization of N-BID did not confer a decoy function, suggesting that it did not successfully compete for the _1C subunit although it shared the BID binding site with the C-BID and BID negative constructs. This lack of dominant-negative effect of N-BID is consistent with previous observations that a short N-terminal isoform of β₁ subunit can modulate the gating of cardiac Ca_V1.2 channels, without promoting membrane trafficking of the channel complex (Cohen et al., 2005). Interestingly, a recent report demonstrated a link between the Ca_V1.2 activity and the cell endocytic machinery. In that study, a reduction in the number of channels present at the plasma membrane was observed when the SH3 domain of the β subunit (also located in the N terminus) was expressed in oocytes (Gonzalez-Gutierrez et al., 2007). The removal of the channels from the membrane by this β₂-SH3 construct is not consistent with our observation that N-BID had no effect on Ca²⁺ currents. This suggests that additional portions of the N terminus of the β subunit may be involved in the regulation of the turnover of the L-type Ca²⁺ channels. Alternatively, it is possible that the spatial cellular localization of N-BID in these vesicle-like compartments may prevent binding interaction with the endogenous _1C protein.

Our study has several limitations. First, the HL-1 cells represent an atrial cell line that expresses robust Ca²⁺ currents and other markers of cardiac function, suggesting that it is a useful model in which to study Ca²⁺ channel regulation (Claycomb et al., 1998; White et al., 2004; Xia et al., 2004). However, the effectiveness of our dominant-negative β₂ constructs clearly may differ between HL-1 cells and native cardiac cells that may express different levels of endogenous β subunits or even process the mutant β subunits differently. Indeed, the success of our β₂ subunit mutants to reduce Ca²⁺ current in cardiac myocytes will depend on the rate of L-type channel turnover in the surface membrane in vivo. The precise turnover rate is unknown, but the fact that changes in Ca²⁺ current in response to norepinephrine or thyroid hormone are evident within 24 to 48 h suggests that the membrane turnover of L-type Ca²⁺ channels is a highly dynamic process (Kim et al., 1987; Maki et al., 1996). Second, we did not attempt to pinpoint the cellular sites involved in the aberrant localization of the C-BID, N-BID, and BID mutants. Further detailed colocalization studies will be needed to identify the cellular compartments or organelles that account for these expression patterns.

Although our initial attempt to detect by immunostaining the endogenous _1C subunit in the HL-1 cells was not successful (Supplemental Figs. A and B), we believe that colocalization of the endogenous _1C subunit with the GFP-tagged β₂ subunit constructs would provide important insight into the mechanism by which these β₂ subunits affect Ca²⁺ channel function. Finally, structure-function studies are needed to optimize the design of dominant-negative β₂ constructs to obtain high-affinity molecules that serve as effective decoys for the L-type Ca²⁺ channels as a prelude to expressing it specifically in heart.

It is noteworthy that new molecular approaches designed to down-regulate L-type Ca²⁺ channel subunits in cardiac myocytes represent alternatives to the traditional use of organic Ca²⁺ channel blocking drugs to attenuate [Ca²⁺]_cyt. The concept of directly titrating the number of Ca²⁺ channel proteins rather than altering the open-state probability of an existing Ca²⁺ channel population may have advantages, particularly if coupled to a cardiac-specific delivery system to provide durability of effect. For example, work by others shows that in vitro gene knockdown of the β₂ subunit using RNA interference technology significantly decreased calcium transients in neonatal rat cardiomyocytes, prevented an increase in relative cell size, and abrogated phenylephrine-induced protein synthesis in a cellular model of hypertrophy (Cingolani et al., 2007). Furthermore, in an aortic-banded rat model of left ventricular hypertrophy, the same authors showed that knockdown of the β₂ subunit was capable of attenuating cardiac hypertrophy without compromising systolic performance, suggesting that the targeted suppression of pathologies involving Ca²⁺ overload may be possible (Cingolani et al., 2007). The adenoviral vectors used in our study are primarily useful in vitro for proof-of-concept, because they are not regarded as ideal clinical vectors due to their immunogenic nature. However, other vectors including adeno-associated viruses containing cardiac-specific promoters may permit targeted knockdown of L-type channels in the heart.

In summary, the data presented in the current study do support the concept that adenovirus-mediated gene transfer of dominant-negative β₂ subunit mutants can effectively and specifically modulate L-type calcium channel expression and function in the HL-1 cell model system of cardiomyocytes.

	Acknowledgements

 
We thank Drs. Zhen Wang and Larisa Buzdugan for excellent technical support, and GibAnn Berryhill for assistance in preparing the manuscript.

	Footnotes

 
This work was supported in part by the University of Arkansas for Medical Sciences (UAMS) Department of Internal Medicine Departmental Research Endowments (to S.T. and J.D.M.), UAMS Department of Pharmacology and Toxicology Research Funds (to J.R.S.), the Department of Veterans Affairs in the form of a VA Merit Award (to J.D.M.), and United States Public Health Service Grants R01 HL-68406 and R01 HL-050818 (to N.J.R.) from the National Institutes of Health (NIH). The University of California Davis/National Institute of Neurological Disorders and Stroke (NINDS)/National Institute of Mental Health (NIMH) NeuroMab Facility (mouse _1C subunit antibody) is supported by NIH Grant U24 NS-050606. The mouse _1C subunit monoclonal antibody (clone L57/46) used in the present study was developed by and/or obtained from the University of California Davis/NINDS/NIMH NeuroMab Facility and maintained by the Department of Pharmacology, School of Medicine, University of California, Davis, CA.

S.T. and S.S. contributed equally to this work.

A preliminary version of some of these results has been presented in abstract form at the following conference: Sonkusare S, Stimers JR, Grain T, Marsh JD, and Télémaque S (2006) Expression of β₂ subunit mutants alters calcium currents in HL-1 cells (FASEB J 20:A1114); Experimental Biology Meeting 2006; 2006 Apr 1–5; San Francisco, CA. American Society for Pharmacology and Experimental Therapeutics, Bethesda, MD.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.128215.

ABBREVIATIONS: Ca_v, Ca²⁺ channel; BID, β interaction domain; SH3, Src homology 3; GK, guanylate kinase; GFP, green fluorescence protein; Full-β₂, full β₂ subunit; Ca_L, L-type Ca²⁺ channel; PCR, polymerase chain reaction; bp, base pair(s); CMV, cytomegalovirus; HEK, human embryonic kidney; I_Ca, Ca²⁺ channel current; I-V, current-voltage; V_1/2, half-maximal activation.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Sabine Télémaque, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham, #832, Little Rock, AR 72205. E-mail: stelemaque{at}uams.edu

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