Caveolin-3 Regulates Protein Kinase A Modulation of the Ca_V3.2 (α_1H) T-type Ca²⁺ Channels

Abstract

Voltage-gated T-type Ca²⁺ channel Ca_v3.2 (α_1H) subunit, responsible for T-type Ca²⁺ current, is expressed in different tissues and participates in Ca²⁺ entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca_v3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca²⁺ in different cells. We examined the localization and regulation of the Ca_v3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca_v3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca_v3.1 and Ca_v3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca_v3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca_v3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca_v3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I_Ca,T) and adenovirus (AdCa_v3.2)-mediated increase in peak Ca_v3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I_Ca,T by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca_v3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca²⁺ signaling during normal and pathological processes.

Introduction

T-type Ca²⁺ channels (TTCC)² are low voltage-activated Ca²⁺ channels, expressed in various tissues including brain and heart and contribute to a variety of physiological functions such as neuronal excitability, hormone secretion, muscle contraction, and pacemaker activity (1–3). Molecular cloning studies have identified three different TTCC isoforms, Ca_V3.1 (α_1G), Ca_V3.2 (α_1H), and Ca_v3.3 (α_1I), which functionally can be distinguished by their electrophysiological properties (4–7). Ca_v3.1 and Ca_v3.2 are the most commonly expressed subunits generating T-type Ca²⁺ current (I_Ca,T) in brain and heart. Ca_v3.2 T-type Ca²⁺ channels are involved in neurological disorders such as epilepsy and pain (8). In the heart, the I_Ca,T participates in Ca²⁺ entry and Ca²⁺-dependent hormonal secretion, pacemaker activity, and arrhythmia (9, 10). The Ca_v3.1 and Ca_v3.2 isoforms are normally expressed in embryonic hearts (11, 12), but postnatal expression of these isoforms diminishes with almost no expression in normal adult ventricular myocytes. However, the TTCCs are re-expressed during conditions of cardiac hypertrophy and heart failure and are reported to be associated with decreased cardiac function (13–15). Ca²⁺ influx through the re-expressed voltage-gated Ca_v3.2 (α_1H) TTCC is indicated to be responsible for inducing pathological cardiac hypertrophy in a pressure overload model (16).

Although we are beginning to understand the physiological role of Ca_v3.2 channels in Ca²⁺ cycling, the precise regulation of these channels has not been defined. To clearly understand the regulation and physiological function of these proteins in normal and diseased stages it is important to define the exact subcellular localization of this protein. Caveolae are specialized membrane microdomains enriched in cholesterol and sphingolipids, contain the signature scaffolding protein caveolins, and provide temporal and spatial regulation of intracellular Ca²⁺ in many cell types (17–19). Three different caveolin isoforms are identified, of which Cav-1 and Cav-2 are ubiquitously expressed (20, 21), whereas Cav-3 is widely expressed in muscle (cardiac, skeletal, and smooth muscle) cells (22) and neuronal tissue (23). A number of ion channels and transporters have been localized to caveolae and associate with Cav-3, including L-type Ca²⁺ channels (Ca_v1.2) (24), the Na⁺ channel (Na_v1.5) (25), pacemaker channels (HCN4) (26), Na⁺/Ca²⁺ exchanger (27), and others (18). Closely associated with these channels are specific macromolecular signaling complexes containing a variety of regulatory proteins including the G protein-coupled receptors and kinases such as PKC and PKA that provide highly localized regulation of the channels (18). The present study therefore was designed to determine the precise subcellular localization and regulation of the Ca_v3.2 subunit of the T-type Ca²⁺ channels. Our results demonstrate that the Ca_v3.2 channel protein is localized to caveolar microdomains in the ventricular myocytes and Cav-3 interacts with Ca_v3.2 channel and regulates its function.

EXPERIMENTAL PROCEDURES

Materials

All chemicals and reagents were procured from Sigma unless otherwise stated. Mouse monoclonal antibodies to Cav-3, cardiac actin, and biotin were obtained from BD Biosciences; rabbit polyclonal antibody to Cav-3 and control IgG antibodies were from Santa Cruz Biotechnology. Rabbit polyclonal antibodies to Ca_v3.1 and Ca_v3.2 channel were from Alamone Labs, Jerusalem, Israel; rabbit polyclonal antibody to Ca_v3.1 was from Millipore. Tetradotoxin was obtained from Calbiochem. PKA inhibitor peptide 14-22, myristoylated (amino acid sequence: Myr-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH₂), was from Sigma.

Cell Culture and Transfection

Wild-type human Ca_v3.1, Ca_v3.2 channels, or Cav-3 protein were expressed in human embryonic kidney 293 (HEK293) cells as previously described (28). The HEK293 cells were maintained in modified DMEM (Invitrogen) and cultured at 37 °C in 5% CO₂. HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen) as described previously (29). 48 h after transient transfection the cells were used for experiments. Neonatal or adult mouse ventricular myocytes were enzymatically isolated as previously described (24, 30). The neonatal myocytes were transfected by the electroporation method by a Nucleofector device (AMAXA) as described earlier (24) using Ingenio electroporation reagent (catalog number MIR 50115) from Mirus Bio, and cells were used for experiments 72–96 h after transfection.

Immunoprecipitation

Isolated neonatal mouse ventricular cardiomyocytes from four 100-mm diameter dishes or adult mouse myocytes (∼2 mg of protein) were rinsed with ice-cold TBS (pH 7.4) and lysed in ice-cold solubilization buffer containing 25 mm Tris-HCl (pH 7.4), 150 mm NaCl, 60 mm n-octyl d-glucoside, 1% Triton X-100, 2 mm phenylmethylsulfonyl fluoride, 5 μg/ml of aprotinin, 5 μg/ml of benzamidin, 5 μg/ml of leupeptin, and 5 μm pepstatin A. The lysate was centrifuged at 10,000 × g for 10 min to remove insoluble debris, and the soluble supernatant was pre-cleared using protein G-Dynabeads (Invitrogen), followed by incubation for 4 h at 4 °C with anti-Cav-3 (5 μg) or anti-Ca_v3.1 or Ca_v3.2 (5 μg) antibodies or control IgG in a total of 450 μl. 50 μl of 1:1 slurry of protein G-Dynabeads was added to the sample and further incubated for 1 h at 4 °C. Beads were washed four times with solubilization buffer on a magnetic stand, and bound proteins were eluted with SDS-PAGE sample buffer by boiling for 5 min. Immune complexes were analyzed by SDS-PAGE (4–15% gradient gels, Bio-Rad) and Western blot by probing with antibodies to Ca_v3.1, Ca_v3.2, and Cav-3.

Cav-3 Subdomain Plasmids, GST-Cav-3 Fusion Plasmids, and Pulldown Assays

Full-length and different subdomains of Cav-3 were generated by standard polymerase chain reaction strategies utilizing the human Cav-3 as template. cDNA clones were constructed of the Cav-3 N terminus domain (amino acids 1–54, Cav-3^1–54), scaffolding domain (amino acids 55–74, Cav-3^55–74), membrane domain (amino acids 75–107, Cav-3^75–107), and C-terminal domain (amino acids 108–151, Cav-3^108–151). The amplified product was cloned into pcDNA3.1 with a TOPO directional cloning system reagent kit from Invitrogen according to the manufacturer's instructions and transformed into Escherichia coli BL21 strain. The plasmid DNA was purified using a Plasmid Maxi kit (Qiagen). DNA sequencing was used to confirm the correctness of the domains. For generations of Cav-3-GST fusion proteins the PCR products were subcloned into Pme1 and Sgf1 sites of pFN2A (GST) Flexi vector (Promega) by restriction digestion. The resulting wild-type and truncated Cav-3 constructs were confirmed by sequencing and transformed into E. coli BL21(DE3) strain. The GST fusion protein was purified from E. coli following induction by 0.1 mm isopropyl 1-thio-β-d-galactopyranoside and linked to glutathione-agarose beads as described earlier (31, 32). For pulldown assays, Ca_v3.2 channel protein was expressed in HEK293 cells, 48 h after transfection the cells were lysed and solubilized on ice using ice-cold solubilization buffer (10 mm Tris, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P40, 1% Triton X-100, 60 mm N-octylglucoside, 2 μm phenylmethylsulfonyl fluoride, 1 μg/ml of aprotinin, 2 μg/ml of benzamidin, 1 μg/ml of leupeptin, and 1 μg/ml of pepstatin A), the lysate was then centrifuged at 10,000 × g for 10 min. The soluble supernatant was collected and then allowed (2 mg of protein) to bind to the MagneGST-agarose beads (Promega) linked with different Cav-3 GST fusion protein constructs. After a 4-h incubation at 4 °C, the sample was washed in 20 mm Tris, 150 mm NaCl, 5 mm EDTA, 0.1% Triton X-100, 0.5% sodium deoxycholate. The eluted sample was then separated and analyzed by SDS-PAGE and Western blot, respectively, by probing with anti-Ca_v3.2 (1:100, rabbit polyclonal, Alomone Labs) and anti-GST antibody (1:500, mouse monoclonal; BD Biosciences).

SDS-PAGE and Western Blot Analysis

Myocyte lysates, or the immune complexes were separated by SDS-PAGE (4–15% gradient acrylamide gel) and transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked by 5% (w/v) dried skim milk in TBS detergent (0.1% Tween 20). Membranes were then probed with specific primary antibodies (Ca_v3.1 or Ca_v3.2, 1:100; Cav-3, 1:1000) followed by a washing step (four times for 10 min each) with TBS detergent. The membranes were then incubated with either goat anti-mouse Ig conjugated to horseradish peroxidase (1:10,000) or goat anti-rabbit IgG to horseradish peroxidase (1:10,000; Bio-Rad) for 1 h and washed (six times for 10 min) with TBS detergent. Immunoreactivity was visualized using peroxidase-based chemiluminescent detection by ECL (Amersham Biosciences).

Biotinylation of Cell Surface Protein and Immunoblotting

Surface biotinylation studies were performed as described earlier (33) in HEK293 cells stably expressing Ca_v3.2 channel protein. Briefly, HEK293 cells stably expressing Ca_v3.2 channels were grown in 100-mm culture dishes and transfected with Cav-3 or GFP. After 48 h, cells were washed three times with PBS and incubated with sulfo-NHS-(LC)-biotin (0.5 mg/ml; Pierce) in PBS at 4 °C for 1 h. Cells were then washed five times with ice-cold PBS to remove residual biotin reagent and solubilized in lysis buffer with protease inhibitors. Lysate proteins were quantified with a bicinchoninic acid assay (Pierce). Proteins (0.5 mg/reaction) were mixed with anti-Cav3.2 antibody (5 μg) immunoprecipitated using the protein G-Dynabeads as described above. The immunoprecipitated sample was analyzed by Western blotting by probing with anti-biotin antibody (mouse monoclonal, 1:5,000, BD Biosciences). Immunoblot membrane was then stripped and re-probed with anti-Ca_v3.2 antibody (1:100) to detect the Ca_v3.2 channel protein signal.

Adenoviral Infection of Ca_v3.2 Channel

Adenoviruses expressing either Ca_v3.2 channel protein (AdCav3.2) or green fluorescent protein (AdGFP) were amplified as described by Brueggemann et al. (34). Isolated neonatal mouse ventricular myocytes were plated on laminin-coated coverslips in a 35-mm Petri dish at 1 × 10⁵ cells and infected with AdCa_v3.2 or AdGFP at a multiplicity of infection of 5–10. More than 90% cells infected with Ad-GFP expressed GFP 48 h after infection. Negligible cell death was observed in cultures infected with adenovirus. Experiments were performed 72–96 h after adenoviral infection.

siRNA-mediated Cav-3 Knockdown

siRNA-mediated knockdown of Cav-3 in isolated neonatal mouse cardiomyocytes was archived by transfecting three pairs of pre-validated Cav-3 specific siRNAs as described earlier (24). Cav-3 sequences targeting different regions for mouse Cav-3 (GenBank^TM accession number NM_007617; sense, GCUUCGACGGUGUAUGGAAtt, and antisense, UUCCAUACACCGUCGAAGCtg; sense, GGUUCCUCUCAAUUCCACCtt, and antisense GGUGGAAUUGAGAGGAACCtc; sense, CGUUCACCGUCUCCAAGUtt, and antisense, UACUUGGAGACGGUGAACGtg) were used for Cav-3 knockdown. Nonspecific siRNA oligos to mouse GAPDH (Applied Biosciences) was used as controls. Briefly, freshly isolated neonatal mouse myocytes were transfected by the electroporation method with the desired oligos (10 nm) and 0.5 μg of cDNA for GFP as previously described (24). Forty-eight to 72 h after transfection myocytes was evaluated for GFP expression and immunostained for Cav-3 (rabbit polyclonal antibody; Santa Cruz Biotechnology 1:500 dilution) and cardiac-specific actin (mouse monoclonal antibody; BD Biosciences; 1:1000 dilution) expression to determine Cav-3 knockdown in the myocytes. The number of GFP-expressing cells divided by the total number of cells in the field of view determined transfection efficiency (data not shown) and was estimated to be between 50 and 60%. Neonatal myocytes expressing GFP fluorescence were used for patch clamp experiments.

Transthoracic Aortic Constriction (TAC)-induced Pressure Overload and Echocardiography Analysis

TAC was performed on 8–12-week-old C57BL/6 male mice as described previously (35). Sham-operated mice were subjected to identical interventions except for the constriction of the aorta. Noninvasive transthoracic echocardiography (35) was performed before and after 4 weeks of TAC surgery in mice, and LV wall thickness, chamber dimension, and contractility were evaluated. The pressure gradients across the aortic constriction were measured to ensure similar pressure overload in the TAC mice.

Immunogold Labeling and Electron Microscopy

Double immunogold labeling co-localization studies and electron microscopy (EM) was performed on isolated and cultured neonatal myocytes as described by Balijepalli et al. (24) using anti-Ca_v3.2 (rabbit polyclonal) and anti-Cav-3 (mouse monoclonal) antibodies and a double silver enhancement technique.

Electrophysiology

Electrophysiological recordings were made from HEK293 cells transiently expressing human cardiac T-type calcium channels (I_Cav3.1 and I_Cav3.2) and Cav-3 as described previously (28). T- and L-type calcium currents were recorded from cultured mouse neonatal cardiomyocytes (3–5 days in vitro). Calcium currents were measured from isolated cells with bright GFP fluorescence with either ruptured or perforated patch configuration. The patch pipettes were pulled from thin walled borosilicate glass capillaries (World Precision Instruments, Inc., Sarasota FL) and filled with an intracellular solution containing the following (in mm) for both HEK293 cells and neonatal cardiomyocytes: 114 CsCl, 10 EGTA, 10 HEPES, 5 MgATP (pH 7.2) adjusted with CsOH. Extracellular buffer for HEK293 cells contained the following (in mm): 128 CsCl, 2 CaCl₂, 1.5 MgCl₂, 10 HEPES, 25 d-glucose (pH 7.4), adjusted with CsOH. For neonatal cardiomyocytes (in mm): 145 tetramethylammonium chloride, 5 CaCl₂, 1 MgCl₂, 5 CsCl, 1,4-aminopyridine, 0.01 tetrodotoxin, 10 HEPES, 5 d-glucose (pH 7.4), adjusted with tetramethylammonium OH. The current-voltage relationship was evoked by step depolarization from −90 to +60 mV with 10-mV step voltage for 200 ms. For perforated patch clamp measurements the pipette solution contained (in mm): 140 Cs-glutamate, 10 HEPES, 0.5 CaCl₂, and 400 μg/ml of amphotericin B (pH 7.2). The external buffer consists of (in mm): 145 tetramethylammonium chloride, 5 CaCl₂, 1 MgCl₂, 10 HEPES, and 5 d-glucose, 5 CsCl, 1 aminopyridine, and tetrodotoxin (1 μm) bath applied. The peak I_Ca,T were measured at a holding potential at −90 mV, a test pulse of −30 mV for 200 ms was applied at 15-s time intervals. After the initial basal I_Ca,T measurement, 3–5-min myocytes were perfused with 100 μm 8-Br-cAMP (in the bath solution) to activate the protein kinase A (PKA)-dependent stimulation of the I_Ca,T. Following stimulation with 8-Br-cAMP, 10 μm nitrendipine and 0.5 mm NiCl were included in the bath solution to block I_Ca,L and I_Ca,T, respectively. In some experiments neonatal myocytes were pretreated with 10 μm myristoylated PKA inhibitor peptide (overnight incubation) to inhibit the PKA activity. The data were collected from a minimum of three different transfections. All experiments were carried out at room temperature with pipette resistance of 1.5–2.5 MΩ. The data were acquired using Axopatch 200B amplifier (Axon Instruments, Foster City, CA) with pCLAMP 10.2. The data were filtered at 5 kHz and digitized at 50 kHz. The current traces were corrected for linear capacitance and leak using -P/4 subtraction protocol.

Data Analysis

Data were analyzed using the Clampfit and Origin 7.5 software programs. The curves for steady state activation and inactivation were fitted using Boltzmann equation. The current-voltage curves were fitted using the Boltzmann function: I = I_max/(1 + exp[− (V − V_1/2)/k]). All the results are presented as the mean ± S.E. and the significance of the observed difference was evaluated by unpaired Student's t test. p value of <0.05 was considered statistically significant.

RESULTS

T-type Ca²⁺ Channel Isoforms Associate with Cav-3 in Ventricular Myocytes and HEK293 Cells

T-type Ca²⁺ channel isoforms Ca_v3.1 and Ca_v3.2 as well as Cav-3 are known to be expressed in neonatal cardiomyocytes. To determine whether Cav-3, Ca_v3.1, and Ca_v3.2 channels are associated in ventricular myocytes, we performed co-immunoprecipitation experiments. Lysates from isolated neonatal mouse ventricular myocytes were solubilized in Triton X-100 and N-octyl d-glucoside-containing buffer and subjected to immunoprecipitation with anti-Cav-3 or control mouse IgG. Western blot analysis shows that Ca_v3.1 and Ca_v3.2 protein bands were detected from the mouse neonatal myocytes lysate (Fig. 1, A and B). Both TTCC isoforms co-immunoprecipitated with Cav-3 from neonatal mouse ventricular myocytes lysates. In a converse experiment we used either anti-Ca_v3.1 or Ca_v3.2 or a control rabbit IgG antibody for immunoprecipitation from neonatal ventricular myocyte lysates and found that Cav-3 co-immunoprecipitated with either of the TTCC isoform antibodies but not with control IgG. These results suggested that the Ca_v3.1 and Ca_v3.2 channels associate with Cav-3 in ventricular myocytes. To further confirm these results in a heterologous expression system we transiently expressed Cav-3 and Ca_v3.1 or Ca_v3.2 channel proteins in HEK293 cells that do not express either of these proteins endogenously. 48 h post-transfection cell lysates were subjected to immunoprecipitation with anti-Cav-3, or control mouse IgG. As demonstrated in Fig. 1C, the T-type Ca²⁺ channel isoform Ca_v3.1 and Ca_v3.2 proteins co-immunoprecipitated with anti-Cav-3 antibody. These results confirmed that the T-type Ca²⁺ channels associate with Cav-3. Non-transfected HEK293 cells did not show protein bands for T-type Ca²⁺ channel isoforms or Cav-3.

FIGURE 1.

T-type calcium channel isoforms Ca_v3.1 and Ca_v3.2 associate with Cav-3. Neonatal mouse myocyte homogenates were subjected to immunoprecipitation with either anti-Ca_v3.1 or Ca_v3.2 or Cav-3 antibodies, and the immunoprecipitates were analyzed by immunoblotting. A, representative immunoblot showing Ca_v3.1 and Cav-3 detected in the immunoprecipitates with either of the two antibodies, whereas control IgG does not immunoprecipitate the proteins. B, representative immunoblot showing Ca_v3.2 and Cav-3 detected in the immunoprecipitates with either of the two antibodies, whereas control IgG does not immunoprecipitate the proteins. C, representative immunoblot showing co-immunoprecipitation of Ca_v3.1 and Ca_v3.2 proteins from transiently expressed HEK293 cell lysates using anti-Cav-3 antibody. Both Ca_v3.1 and Ca_v3.2 channel proteins co-immunoprecipitate with anti Cav-3 antibody, whereas control IgG does not immunoprecipitate either protein. Signal for Ca_v3.1 and Ca_v3.2 channel proteins was not detected in the untransfected HEK293 cell lysates. Results are representative of 4 different experiments.

Ca_v3.2 Channel Protein Co-localized with Cav-3 Relative to Caveolae in Ventricular Myocytes

To determine the precise localization of Ca_v3.2 channels we used the immunogold labeling technique combined with electron microscopy. To determine localization of the Ca_v3.2 channels, we initially used isolated mouse neonatal ventricular myocytes that were fixed and immunogold co-labeled with anti-Cav-3 and anti-Ca_v3.2 antibody. Transmission electron micrographs revealed two distinct populations of different sized gold particles (Fig. 2) present on surface membrane invaginations typical of caveolae. The small gold particles identify anti-Cav-3 labeling (arrows) and large gold particles (as a result of double silver enhancement, arrowhead) identify anti-Ca_v3.2 channel protein labeling suggesting the co-localization of the Ca_v3.2 with Cav-3 relative to caveolae (Fig. 2, a and c). Immunogold labeling showed labeling for Cav-3 but did not show Ca_v3.2 channel staining in the ventricular myocytes of normal mouse adult heart tissue as, cardiac T-type Ca²⁺ channel isoforms are normally expressed during development but not expressed in the adult hearts (Fig. 2, e and g). However, these channels are known to re-express during pressure overload-induced cardiac hypertrophy and heart failure (11–15). Adult mice were subjected to the TAC procedure to generate pressure overload-induced cardiac hypertrophy. Echocardiography analysis (supplemental Table S1) confirmed induction of cardiac hypertrophy. Adult hearts were fixed by perfusion fixation and co-immunogold labeling was performed. As shown in the transmission electron micrograph (Fig. 2, b and d), Ca_v3.2 channel protein (large gold particle) and Cav-3 (small gold particle) were co-localized relative to caveolae. Also, the gold particle distribution was restricted to sarcolemma regions in the ventricular myocytes suggesting specific caveolar localization of the protein. In control samples (hypertrophic adult mouse heart) from which the primary antibodies had been omitted, did not show gold particle staining.

FIGURE 2.

Ca_v3.2 and Cav-3 are colocalized relative to caveolae in mouse ventricular myocyte. Representative transmission electron micrographs show immunogold labeling for Ca_v3.2 (large particle, arrowhead) and Cav-3 (small particle, arrow) in neonatal cardiomyocyte (a and c are enlarged) and hypertrophic adult ventricular myocytes (b and d are enlarged). Normal adult ventricular myocytes show staining for Cav-3 but not for Ca_v3.2 (e and g are enlarged). Control image in f (portion enlarged in h) shows no primary antibody from hypertrophic adult ventricular myocyte. Scale bars, 100 nm.

Co-expression of Cav-3 Inhibits I_Cav3.2 but Not I_Cav3.1 in HEK293 Cells

To determine the functional impact of Cav-3 association on T-type Ca²⁺ channel currents we used a heterologous system of HEK293 cells, which provides a convenient expression system to study specific transfected T-type Ca²⁺ channels and Cav-3, given the lack of endogenous expression of these proteins. HEK293 cells have been used by us and others to study the heterologous expression of T-type Ca²⁺ channels and a wide array of other ion channels (28, 29, 36–39). Human TTCC isoforms Ca_v3.1 and Ca_v3.2 were separately co-expressed with Cav-3 in HEK293 cells and the respective currents were measured using the whole cell patch clamp technique. Currents were measured from a holding potential of −90 mV and stepped to 60 mV in 10-mV increments for 200 ms (as shown in the Fig. 3, B and inset D). Fig. 3, B and D, shows the current-voltage relationship of I_Cav3.1 and I_Cav3.2, respectively, with and without co-expression of Cav-3. Co-expression of Cav-3 did not effect the I_Cav3.1 (-32.8 ± 5 pA/pF, n = 11) compared with expression of Ca_v3.1 with GFP (−33.48 ± 4 pA/pF, n = 11). On the other hand co-expression of Cav-3 with Ca_v3.2 significantly reduced I_Cav3.2 (−11.48 ± 3 pA/pF, n = 11) compared with Ca_v3.2 + GFP (−31 ± 4 pA/pF, n = 11). We also investigated the effect of co-expression of Cav-3 on the biophysical properties of the I_Cav3.2 in HEK293 cells. Co-expression of Cav-3 did not affect Ca_v3.2 channel activation and inactivation properties. These data are presented in supplemental Fig. S1.

FIGURE 3.

Co-expression of Cav-3 inhibits I_Cav3.2 but not I_Cav3.1 in HEK293 cells. Whole cell voltage clamp recordings of T-type Ca²⁺ were performed in HEK293 cells expressing either Ca_v3.1 or Ca_v3.2 alone or co-expressing with Cav-3, by using a holding potential of −90 mV and step pulsed to 60 at 10 mV as shown in the inset. A, representative whole cell Ca²⁺ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca_v3.1 alone and Ca_v3.1 with Cav-3. B, average current density plotted against the change in test potential for Ca_v3.1 (■) alone and with Ca_v3.1 + Cav3 (●). C, representative whole cell Ca²⁺ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca_v3.2 alone and Ca_v3.2 with Cav-3. D, average current density plotted against the change in test potential for Ca_v3.2 (■) alone and with Ca_v3.2 + Cav-3 (●). Data represents mean ± S.E. (n = 11 from 4 different transfections). Co-expression of Cav-3 does not alter plasma membrane expression of Ca_v3.2 channel protein. HEK293 cells were transfected with cDNAs of Ca_v3.2 + GFP or Ca_v3.2 + Cav-3 or GFP alone. E, cell lysates were precipitated with neutravidin beads and the sample was analyzed by Western blots by probing with anti-Ca_v3.2 or anti-β-actin antibodies as indicated in the representative immunoblot. Similar signal intensity for the biotinylated Ca_v3.2 protein was detected with Ca_v3.2 + GFP or Ca_v3.2 + Cav-3. F, a portion of the total lysate (50 μl) sample as input from 3 groups was also analyzed by probing with anti-Ca_v3.2 and anti-β-actin for loading control. Similar signal intensity for the Ca_v3.2 channel protein was detected with either Ca_v3.2 alone or with co-expression of Cav-3 and similar β-actin signal was detected between the three groups of cells demonstrating identical sample loading. Data are representative of three different experiments.

Effect of Co-expression of Cav-3 on Plasma Membrane Expression of Ca_v3.2 Channel Protein

To understand the mechanism of Cav-3 inhibition of the I_Cav3.2 we investigated if Cav-3 co-expression alters trafficking and reduces the surface expression of the Ca_v3.2 channels. One way to determine the number of channels expressed on the plasma membrane is to measure the gating currents, however, given the small I_Cav3.2 amplitudes (300–500 pA) we could not measure the gating currents to estimate the number of plasma membrane-expressed Ca_v3.2 channels with Cav-3 co-expression. We used an alternative approach of cell surface biotinylation of the Ca_v3.2 channels. HEK293 cells were transfected with cDNAs of Ca_v3.2 + GFP or Ca_v3.2 + Cav-3, or GFP alone. Cell lysates were precipitated with neutravidin beads and samples were analyzed by Western blots by probing with anti-Ca_v3.2 or anti-β-actin antibody. As shown in a representative Western blot (Fig. 3E), no difference was noticed in the biotinylated Ca_v3.2 protein signal intensity when Ca_v3.2 was expressed alone (Ca_v3.2 + GFP) or co-expressed with Cav-3 (Ca_v3.2 + Cav-3) suggesting that co-expression of Cav-3 did not affect the surface membrane expression of Ca_v3.2 channel. These experiments were repeated three times and the signal for the biotinylated Ca_v3.2 band was semi-quantitatively estimated by densitometry. We found that the mean signal density for the biotinylated Ca_v3.2 band was not different between groups (data not shown). The Ca_v3.2 protein signal was absent in the sample that was transfected with GFP alone. We did not detect a signal for β-actin, demonstrating the biotinylation of only surface membrane proteins. The signal for Cav-3 as also absent in the neutravidin pulldown as Cav-3 is localized to the inner leaflet of the plasma membrane bilayer and not biotinylated. A portion of the lysate sample from 3 groups was also analyzed by probing with anti-Ca_v3.2 and anti-β-actin (Fig. 3, panel F) for loading control. Again similar signal intensity for the Ca_v3.2 channel protein was detected with either Ca_v3.2 alone or with co-expression of Cav-3 and a similar β-actin signal was detected between the three groups of cells demonstrating identical sample loading.

Ca_v3.2 Interaction Site on Cav-3

We next examined the site of interaction for Cav-3 with the Ca_v3.2 channel protein. We created five different GST fusion constructs of Cav-3 based on known domains (illustrated in Fig. 4A and see Ref. 18): 1) full-length (Cav-3FL), 2) Cav-3Nterm (Cav-3^1–54), 3) Cav-3Scaf (Cav-3^55–73), 4) Cav-3Mem (Cav-3^74–106), and 5) Cav-3Cterm domain (Cav-3^107–151) using a bacterial system as described under “Experimental Procedures.” Ca_v3.2 channel protein was expressed in HEK293 cells, and the lysates were incubated with GST alone or different GST-Cav-3 fusion proteins linked to glutathione beads. Following that procedure, a pulldown assay was performed and samples were analyzed by Western blotting. As demonstrated in Fig. 4B, the Ca_v3.2 channel was found to interact and associate with the full-length GST fusion protein of Cav-3 (GSTCav-3FL) and N terminus GST fusion protein of Cav-3 (GST-Cav3NT; amino acids 1–54) but not associate with GST-Cav-3Scaf (Cav-3^55–73), GST-Cav-3Mem (Cav-3^74–106), and the GST-Cav-3C-terminus domain (Cav-3^107–151). This suggested that the Ca_v3.2 subunit interacts with Cav-3 and specifically interacts with the N-terminal region of the Cav-3.

FIGURE 4.

N terminus region of Cav-3 interacts with Ca_v3.2 channel protein and inhibits I_Cav3.2. A, diagram of the domain structure of Cav-3. GST was fused with full-length Cav-3 and the different Cav-3 domains and tested for an ability to interact with the Ca_v3.2 channel. B, representative Western blot analyses of the GST pulldown assay. Ca_v3.2 channels were expressed in HEK293 cells, and lysates were incubated with different GST-Cav-3 fusion proteins as described under “Experimental Procedures”: full-length (GST-Cav-3FL), Cav-3 N terminus (GST-Cav-3NT), Cav-3 scaffolding domain (GST-Cav-3Scaf), Cav-3 membrane domain (GST-Cav-3Memb), Cav-3 C terminus domain (GST-Cav-3CT), or GST alone. The pulldown samples were analyzed by probing with anti-GST and anti-Ca_v3.2 antibodies. C, HEK cells were transiently expressed with Ca_v3.2 alone or Ca_v3.2 with different Cav-3 domains (Cav-3NT, Cav-3Scaf, Cav-3CT, and Cav-3Memb). I_Cav3.2 density was measured by the whole cell patch clamp technique and plotted against a change in test potentials. Data represent mean ± S.E. (n = 7 each, *, p > 0.005 with respect to control) from three different transfections.

Co-expression of Cav-3 N Terminus Domain Inhibits I_Cav3.2 in HEK293 Cells

Our GST pulldown assay demonstrates that the N terminus of Cav-3 interacts with the Ca_v3.2 channel. To determine the functional impact of this interaction on Ca_v3.2 channels, we generated cDNA constructs of the N terminus region (Cav-3^1–54) and other regions of Cav-3 as described under “Experimental Procedures.” These cDNAs along with the Ca_v3.2 cDNA were then transiently transfected into HEK293 cells. I_Cav3.2 was then measured by whole cell patch clamp analysis. As demonstrated in Fig. 4B, co-expression of Cav-3Nterm significantly reduced the I_Cav3.2 (11.7 ± 1 pA/pF, n = 7) compared with Ca_v3.2 alone (24.1 ± 2 pA/pF, n = 7). On the other hand co-expression of Cav-3Cterm, Cav-3Scaf, or Cav-3Memb with Ca_v3.2 channels did not alter the I_Cav3.2. These data further demonstrate that interaction of the N terminus region of Cav-3 with the Ca_v3.2 channel protein modulates the channel function.

Overexpression of Cav-3 Inhibits I_Ca,T in Mouse Neonatal Ventricular Myocyte

Our data from the heterologous expression system of HEK293 cells demonstrated that co-expression of Cav-3 specifically inhibited the I_Cav3.2. Next we investigated if Cav-3 will have a similar effect on the native T-type Ca²⁺ current (I_Ca,T) using isolated neonatal cardiomyocytes where Cav-3 and the Ca_v3.2 channel isoform are endogenously expressed. Neonatal ventricular myocytes express both isoforms of TTCC as well as Ca_v1.2, L-type Ca²⁺ channels. T-type Ca²⁺ channels can be distinguished from L-type Ca²⁺ channels on the basis of their conductance and gating properties (40). The TTCCs are known to activate at significantly more negative membrane voltage potentials with a threshold for activation of I_CaT about −60 mV and peak I_CaT between −30 mV at physiological Ca²⁺ concentrations (1, 9, 16), whereas the Ca_v1.2 channels begin to activate at about −30 mV and the peak I_Ca,L is elicited at more positive potentials (10 mV) (24). To isolate the pure I_Ca,T from high voltage-activated L-type currents we used a dual pulse protocol as described earlier (16). Using a whole cell patch clamp technique (Fig. 5) the total I_Ca (I_Ca,Tot) was recorded from myocytes using a holding potential of −90 mV and pulsed to 60 mV in 10-mV steps for 200 ms, followed by a brief holding potential of −50 mV and further pulsed to 70 mV in a 10-mV steps for 200 ms to record the I_Ca,L. To obtain the I_Ca,T, traces of I_Ca,L (holding potential −50 mV; Fig. 5, B and C) were subtracted from the corresponding trace of I_Ca,Tot with a holding potential of −90 mV. Thus we found that peak I_Ca,T was at −30 mV in these cells. Cav-3 or GFP alone were overexpressed in the isolated mouse neonatal ventricular myocytes by electroporation as described earlier (24). Using the same dual pulse protocol we measured the I_Ca,Tot, I_Ca,L, and then obtained I_Ca,T by subtraction as demonstrated in Fig. 5, and found that the average peak I_Ca,T density for nontransfected cells (−5 ± 0.7 pA/pF, n = 11) or GFP-transfected cells (−4.7 ± 1 pA/pF, n = 11) was similar (Fig. 5E). On the other hand overexpression of Cav-3 significantly reduced the I_Ca,T 42% (−2.1 ± 1 pA/pF, n = 8) compared with GFP-transfected control cells (Fig. 5, D and E). Overexpression of Cav-3 had no impact on the average peak I_Ca,L density (Fig. 5F).

FIGURE 5.

Overexpression of Cav-3 inhibits I_Ca,T in isolated mouse neonatal ventricular myocytes. Whole cell patch clamp analysis was performed in neonatal myocytes transfected with Cav-3 or GFP (control) using a protocol as described under “Results” and shown in the inset on top. A and C show representative current traces of GFP and Cav-3-transfected cells, respectively, using a holding potential of −90 mV (left) and −50 mV (middle) to a step depolarization to the respective indicated command potential. The traces on the right are obtained by subtracting the middle trace from the left current trace and represent the T-type Ca²⁺ current. Panels C and D, representative current density plot generated by a series of step depolarization potentials of 10 mV at different holding potentials, i.e. −90 mV (I_Ca,Total, ■) and −50 mV (I_Ca,L, ●) the difference is the T-type current (I_Ca,T, ▴) in control (GFP) and Cav-3 overexpression cells, respectively. E, peak current density of I_Ca,T with or without overexpression of Cav-3. F, peak I_Ca,L densities with and without overexpression of Cav-3. The data represent mean ± S.E. (n = 8–11 cells, *, p > 0.05) from 3 different transfections.

Neonatal ventricular myocytes express Ca_v3.1 and Ca_v3.2 and kinetic properties of I_Cav3.1 and I_Cav3.2 closely resemble native I_CaT. Thus it is difficult to differentiate between the two different current components in a native cardiomyocyte system based on their biophysical properties. Studies have shown that I_Cav3.1 and I_Cav3.2 can be differentiated by their sensitivity to block by Ni²⁺. Recombinant Ca_v3.2 channel is shown to be blocked by low concentrations of Ni²⁺ (IC₅₀ = 12 μm), whereas the Ca_v3.1 channel is blocked by higher concentrations of Ni²⁺ (IC₅₀ = 250 μm) (41). However, in our conditions we did not find it practical to use Ni²⁺ to differentiate between the components of TTCCs because a higher concentration of Ni²⁺ is needed to block I_Cav3.1. So we used an adenoviral overexpression model of I_Cav3.2 and studied the effect of Cav-3 on the I_Ca,T. Isolated neonatal myocytes were transfected with either Cav-3 or GFP alone as above and then infected with AdCa_v3.2 or AdGFP. Overexpression of Ca_v3.2 proteins was confirmed by Western blot analysis (data not shown) and by measuring I_Cav3.2. AdCa_v3.2-treated cells showed significantly increased I_Cav3.2 (-32.4 ± 7 pA/pF) compared with AdGFP-treated control cells (−4.9 ± 1 pA/pF) or non-treated control cells (Fig. 6A). Co-expression of Cav-3 significantly reduced (89%) the AdCa_v3.2-induced I_Cav3.2 (−4.37 ± 1pA/pF), suggesting that Cav-3 inhibits and regulates that I_Cav3.2 in neonatal cardiomyocytes. AdCa_v3.2 or AdGFP treatment did not alter the average I_Ca,L density in the neonatal ventricular myocytes (Fig. 6B).

FIGURE 6.

Cav-3 inhibits I_Cav3.2 in mouse neonatal ventricular myocytes. A, average peak I_Ca,T density in neonatal ventricular myocytes infected with AdGFP control or with either AdCa_v3.2 or AdCa_v3.2+Cav-3 as indicated in the bar plot. Cav-3 overexpression significantly inhibited the AdCa_v3.2-mediated increased I_Cav3.2. B, average densities of I_Ca,L in neonatal ventricular myocytes infected with adenovirus as indicated. Adenovirus infection did not alter the average I_Ca,L density in the cells. The data represent mean ± S.E. (n = 3–5 cells, *, p > 0.005 with respect to AdCa_v3.2). Whole cell patch clamp analysis on the effect of siRNA-mediated knockdown of Cav-3 on I_Ca,T in mouse neonatal ventricular myocytes. C, average peak I_Ca,T density from nontransfected control (NT), GFP control, siRNA to Cav-3 or siRNA to GAPDH (control)-transfected cells as indicated in the bar plot. Representative corresponding T-type current traces are shown above. D, average densities of I_Ca,L in neonatal ventricular myocytes transfected as above and representative corresponding I_Ca,L current traces are shown above the bar plot. The average peak I_Ca,T or I_Ca,L was not significantly different and the data represent mean ± S.E., number of cells used are indicated in parentheses.

To determine the involvement of Cav-3 on the modulation of Ca_v3.2 channels in neonatal cardiomyocytes, we investigated the impact of the specific inhibition of Cav-3 expression using siRNA-mediated gene silencing as described earlier (24). Three different siRNA oligos specific to mouse Cav-3 or control siRNA to GAPDH were co-transfected with GFP into isolated mouse neonatal cardiomyocytes. 72 h after transfection knockdown of Cav-3 was established by Western blot analysis and immunofluorescence imaging for Cav-3, which confirmed that Cav-3 siRNA-transfected cells (GFP-expressing) exhibited near complete knockdown of Cav-3 (see supplemental Fig. S2). Whole cell electrophysiology was performed on GFP expressing cells (green fluorescence) to measure I_Ca,T. siRNA-mediated knockdown of Cav-3 did not alter the I_Ca,T densities (−4.5 ± 0.8 pA/pF n = 22), and was similar to control (GAPDH) siRNA-transfected myocytes (−5.4 ± 1 pA/pF, n = 21) or nontransfected neonatal cardiomyocytes (5 ± 0.7 pA/pF, n = 9) (Fig. 6C). siRNA-mediated knockdown of Cav-3 also had no effect on the average I_Ca,L density as shown in Fig. 6D. A similar effect of siRNA-mediated knockdown on the I_Ca,L was observed in an earlier study from our group when siRNA-mediated knockdown of Cav-3 did not alter the mean I_Ca,L current densities compared with control siRNA-treated neonatal ventricular myocytes (24).

siRNA-mediated Knockdown of Cav-3 Eliminates Protein Kinase A Regulation of the I_Ca,T in Mouse Neonatal Myocytes

Overexpression of Cav-3 inhibited the I_Cav3.2 but the siRNA-mediated inhibition of Cav-3 expression did not affect the I_Ca,T density in neonatal myocytes. We reasoned that Cav-3 may play an important role in regulation of Ca_v3.2 channels and Cav-3 knockdown may alter regulation of the I_Ca,T in neonatal mouse cardiomyocytes. The I_Ca,T and I_Cav3.2 are reported to be augmented by cAMP-dependent protein kinase (PKA) (42–44). We investigated the PKA regulation of the I_Ca,T in neonatal myocytes using 8-bromo-cAMP (membrane permeable cAMP-a known activator of PKA) by perforated clamp analysis. Initially a dose-dependent stimulation of I_Ca,T was performed with 8-Br-cAMP and we found a maximal stimulation of I_Ca,T at 100 μm concentrations (data not shown). In the myocytes that were transfected with control siRNA, 8-Br-cAMP resulted in a PKA-mediated increased stimulation of the peak I_Ca,T to 126 ± 6% (Fig. 7, A and D). 8-Br-cAMP is also known to stimulate I_Ca,L through activation of the high voltage Ca_V1.2 channels in ventricular myocytes at a much higher concentration (1 (45) and 2 mm (46)). To eliminate the possible involvement of I_Ca,L in the 8-Br-cAMP-induced I_Ca response, the cells were perfused with 10 μm nitrendipine, a specific blocker of the I_Ca,L. Perfusion with 10 μm nitrendipine did not affect the peak I_Ca,T. However, upon perfusion with 0.5 mm NiCl, the I_Ca,T was completely abolished, suggesting that 8-Br-cAMP stimulation of the I_Ca is specifically through activation of the T-type Ca²⁺ channel. To confirm that 8-Br-cAMP stimulation of the I_Ca,T is dependent on PKA, we pretreated neonatal myocytes (8–12 h incubation) with the myristoylated PKA inhibitor peptide fragment 14-22 (10 μm). Pretreatment of cells with the specific PKA inhibitor peptide failed to evoke 8-Br-cAMP stimulation of the I_Ca,T in the control siRNA-transfected myocytes, suggesting a PKA-dependent augmentation of I_Ca,T by 8-Br-cAMP (Fig. 7, C and D). In the next set of experiments, we used myocytes that were transfected with siRNA to Cav-3. In contrast with the control siRNA-treated cells the siRNA-mediated Cav-3 knockdown almost completely abolished 8-Br-cAMP stimulation of the I_Ca,T in the myocytes (Fig. 7, B and D). These findings confirm that Cav-3 is required for PKA-mediated regulation of the Ca_v3.2 T-type Ca²⁺ channels in the mouse neonatal ventricular myocytes.

FIGURE 7.

siRNA-mediated knockdown of Cav-3 expression eliminated PKA-mediated 8-Br-cAMP stimulation of I_Ca,T in neonatal mouse ventricular myocytes. Perforated patch whole cell voltage clamp recordings of I_Ca,T were performed by using a holding potential of −90 mV with 50-ms test pulses to −30 mV every 15 s in myocytes. A, peak I_Ca,T is increased by stimulation with 8-Br-cAMP (100 μm) in a representative control siRNA-treated myocyte (whole cell capacitance = 8.6 picofarads); B, siRNA-mediated Cav-3 inhibition eliminated 8-Br-cAMP stimulation of I_Ca,T in a representative myocyte (whole cell capacitance = 7.9 picofarads). C, pretreatment with 10 μm PKA inhibitor peptide completely inhibited the 8-Br-cAMP stimulation of the I_Ca,T in a representative control siRNA-treated myocyte (whole cell capacitance = 11.9 picofarads). In all the groups of cells, perfusion with 10 μm nitrendipine (Nitrend) did not block I_Ca, whereas 0.5 mm Ni²⁺ completely inhibited I_Ca, indicating a stimulation of the T-type Ca²⁺ current by 8-Br-cAMP. D, average effect of 8-Br-cAMP stimulation on I_Ca,T in myocytes with and without Cav-3 siRNA inhibition. The data represent mean ± S.E., number of cells used is indicated in parentheses *, p < 0.001 relative to control.

DISCUSSION

In the present study we describe regulation of the Ca_v3.2 subunit of T-type Ca²⁺ channels by Cav-3 in the mouse ventricular myocytes. Double immunogold labeling and electron microscopy imaging data clearly demonstrate co-localization of the Ca_v3.2 channel with Cav-3 relative to caveolae in the mouse ventricular myocytes. Co-immunoprecipitation analysis suggested an association of Ca_v3.1 and Ca_v3.2 channel isoforms with Cav-3, the GST pulldown assay confirmed a close interaction between the Ca_v3.2 channel protein and Cav-3. Functional analysis using whole cell patch clamp in the heterologous expression system of HEK293 cells demonstrated Cav-3 inhibition of the Ca_v3.2 channel but not Ca_v3.1 channel. In addition, we show that the N terminus region of Cav-3 interacts with the Ca_v3.2 channel and modulates the I_Cav3.2. Whole cell patch clamp studies using the neonatal cardiomyocytes demonstrated specific inhibition of I_Cav3.2 by Cav-3. On the other hand, siRNA-mediated knockdown of Cav-3 eliminated PKA regulation of the I_Ca,T.

T-type Ca²⁺ channels are known to modulate Ca²⁺ influx, membrane potential and hormone secretion. Of the three different T-type Ca²⁺ channel isoforms reported, alternative splicing of the Ca_v3.2 channels results in functional diversity of the channel during cardiac development (47). Recent findings using a heterologous expression system of HEK293 cells suggest a complex regulation for the Ca_v3.2 channels. It is well known that G-protein-dependent signaling pathways modulate native T-type Ca²⁺ channels (2). Extensive studies from the laboratory of P. Berrett have demonstrated Ca_v3.2 channel regulation by PKA (48), Ca²⁺/calmodulin-dependent protein kinase II (49, 50), and inhibition by Gβγ (51, 52). Another study demonstrated a selective inhibition of the Ca_v3.2 channel by corticotropin releasing factor receptor-1 is likely mediated by βγ (53). On the other hand, a recent study demonstrated phospholipase Cβ, and protein kinase C modulation of the Ca_v3.2 channel mediated by G_q/11 in a voltage and Gβγ independent fashion (54). In addition in DRG neurons, monocyte chemoattractant protein-1 (a cytokine) was shown to inhibit Ca_v3.2 channels (55). The present work demonstrates that Cav-3 modulates transiently expressed Ca_v3.2 in HEK293 cells and native Ca_v3.2 current in neonatal mouse cardiomyocytes. Co-expression of Cav-3 inhibited I_Cav3.2 without altering the biophysical properties of the Ca_v3.2 channel, suggesting that Cav-3 modulates Ca_v3.2 current possibly in a voltage independent fashion. Our surface biotinylation data demonstrate that Cav-3 co-expression did not alter plasma membrane expression of the Ca_v3.2 channels, which rules out the possibility of internalization of the Ca_v3.2 channels. Thus, Cav-3 inhibition of I_Cav3.2 could possibly result through different signaling molecules associated with Cav-3.

Although it is clear from our GST-Cav-3 pulldown experiments that the N terminus region of Cav-3 interacts with the Ca_v3.2 channel and modulates the I_Cav3.2, there exists a likely possibility of direct interaction between the Ca_v3.2 channel and Cav-3. The intracellular linker region between domains II and III for the Ca_v3.2 channel contains clusters of serine and threonine residues and has been identified as an important region for interaction and regulation of the channel by serine/threonine kinases and G-protein signaling pathway proteins (3). In this context, it may not be unreasonable to hypothesize an interaction between the N terminus region of Cav-3 and the intracellular loop between domains II and III of the Ca_v3.2 channel. Further studies are needed to identify the site of Ca_v3.2 channel involved in the interaction with Cav-3, which may also help us understand dynamic regulation of the Ca_v3.2 channels.

We observed that siRNA-mediated knockdown of Cav-3 did not affect the I_Ca,T density in the mouse neonatal cardiomyocytes but abolished PKA regulation of the I_Ca,T. Similar to this observation, in an earlier study we had demonstrated that a subpopulation of the Ca_v1.2 L-type Ca²⁺ channels are localized to caveolae and siRNA-mediated Cav-3 knockdown did not affect the I_Ca,L density in neonatal ventricular myocytes. However, Cav-3 knockdown specifically inhibited the β₂-adrenergic receptor regulation of the Ca_v1.2 channels (24). Caveolins contain scaffolding domains that interact with multiple signaling molecules including G-proteins and coupled receptors, regulatory proteins such as Ca²⁺/calmodulin-dependent protein kinase II, PKA, and PKC, and some ion channels, thereby providing temporal and spatial regulation of cellular signal transduction (18, 56, 57). The subcellular localization of ion channels to caveolae allows the integration of these channels into specific macromolecular signaling complexes in a distinct lipid microenvironment providing for their precise modulation. Caveolar localization and Cav-3 inhibition of Ca_v3.2 channels in neonatal cardiomyocytes explains the compartmentalized and dynamic regulation of the Ca_v3.2 channels by PKA and possibly other kinases in the native cells. Perhaps, caveolar localization and Cav-3 interaction of the Ca_v3.2 are necessary for precise modulation of this channel not only by PKA but also by different signaling molecules during normal and pathological states.

Expression of Cav-3 is developmentally regulated as it increases postnatally and reaches maximum levels by days 4–5 followed by a decrease to stable expression levels seen in the adult cardiomyocytes (58, 59). The cardiac T-type Ca²⁺ channels are abundantly expressed during cardiac development but their expression is not detected in normal adult ventricular myocytes. It is well established that moderate to severe ventricular tissue remodeling occurs, with associated changes at the single myocyte level, during pathological states such as cardiac hypertrophy and heart failure (60–62). During cardiac hypertrophy, Ca_v3.1 and Ca_v3.2 channels are known to be up-regulated in the ventricular myocytes. Caveolae and expression of Cav-3 is also known to be altered during these conditions (63, 64). It may also be possible that during the ventricular remodeling process altered Cav-3 and caveolae expression could alter subcellular localization and expression of the Ca²⁺ channels, including the Ca_v3.2 channels that may result in altered coupling and regulation of the channels. The Ca_v3.2 T-type Ca²⁺ channels are involved in various diseases such as epilepsy (39, 65–67), pain (68), hypertension (69), and cardiac hypertrophy (16). In fact, using genetic deletion of CACNA1H, which encodes the Cav3.2 channel, it was demonstrated that the Ca_v3.2 T-type Ca²⁺ channel is required for induction of pathological cardiac hypertrophy (16). One other report indicated that overexpression of Cav-3 is protective against agonist-induced hypertrophic responses in the rat neonatal cardiomyocytes (70). In our studies overexpression of Cav-3 specifically inhibited the T-type Ca²⁺ current without affecting the L-type Ca²⁺ currents, which are also known to localize to caveolae and associate with Cav-3 (24). Our data also demonstrate that Cav-3 specifically inhibited the adenovirus-mediated increased overexpression of I_Cav3.2. In this context our finding may have important therapeutic implications pertaining to the T-type Ca²⁺ channel block, because, T-type Ca²⁺ channel blockers have been proposed to be useful in the therapeutics of a variety of conditions including hypertension and heart failure (69, 71). Further studies are needed to understand the role of Cav-3 and caveolae in the ventricular remodeling process during cardiac disease conditions such as hypertrophy and heart failure. Regulation of the T-type Ca²⁺ channels and Ca²⁺ signaling by caveolae is important for understanding the mechanism of pathological changes in cardiac hypertrophy and heart failure.

In summary, our results demonstrate for the first time a precise caveolar localization of the Ca_v3.2 T-type Ca²⁺ channel in cardiomyocytes. We show that Cav-3 interacts with the Ca_v3.2 channels and modulates PKA regulation of I_Ca,T in the ventricular myocytes. Our studies provide the basis for understanding the compartmentalized regulation of the T-type Ca²⁺ channel in cardiomyocytes and other cell types in normal and pathological conditions.