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Am J Physiol Heart Circ Physiol 291: H1614-H1622, 2006; doi:10.1152/ajpheart.00095.2006
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Protein kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine 81 phosphorylation site

Ghayath Baroudi,1 Yongxia Qu,1 Omar Ramadan,1 Mohamed Chahine,2 and Mohamed Boutjdir1,3

1Department of Cardiovascular Research, Veterans Affairs New York Harbor Healthcare System and Departments of Physiology and Pharmacology, Anatomy and Cell Biology, and Medicine, State University of New York Downstate Medical Center, Brooklyn, New York; 2Québec Heart Institute, Laval Hospital and Department of Medicine, Laval University, Sainte-Foy, Quebec, Canada; and 3Department of Medicine, New York University School of Medicine, New York, New York

Submitted 23 January 2006 ; accepted in final form 18 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Cav1.3 ({alpha}1D) variant of L-type Ca2+ channels plays a vital role in the function of neuroendocrine and cardiovascular systems. In this article, we report on the molecular and functional basis of {alpha}1D Ca2+ channel modulation by protein kinase C (PKC). Specifically, we show that the serine 81 (S81) phosphorylation site at the NH2-terminal region plays a critical role in {alpha}1D Ca2+ channel modulation by PKC. The introduction of a negatively charged residue at position 81, by converting serine to aspartate, mimicked the PKC phosphorylation effect on {alpha}1D Ca2+ channel. The modulation of {alpha}1D Ca2+ channel by PKC was prevented by dialyzing cells with a 35-amino acid peptide mimicking the {alpha}1D NH2-terminal region comprising S81. In addition, the data revealed that only betaII- and {epsilon}PKC isozymes are implicated in this regulation. These novel findings have significant implications in the pathophysiology of {alpha}1D Ca2+ channel and in the development of PKC isozyme-targeted therapeutics.

calcium channels; protein kinase c isozymes; patch clamp

L-TYPE VOLTAGE-GATED Ca2+ channels (VGCC) are widely expressed in various tissues and exert pivotal functions in excitation-contraction coupling in muscles, hormone secretion in endocrine cells, gene expression and regulation in neurons, and other cellular functions. Four types of L-type VGCC have been described (4). Whereas {alpha}1S and {alpha}1F are mainly restricted to skeletal muscle and retinal neuron, respectively (1, 4, 16, 22), the {alpha}1C is expressed in the heart, vascular smooth muscles, and neurons (10, 32). More recently, {alpha}1D, generally thought to be restricted to neuroendocrine cells (5, 7, 10, 15, 29, 33, 37), was also identified in the heart (20, 26, 28, 39). In this regard, {alpha}1D VGCC knockout mice exhibited sinus bradycardia and atrioventricular block (20), and {alpha}1D has been implicated in the pathophysiology of autoimmune-associated heart block (27).

The protein kinase C (PKC) signaling pathway plays a central role in the regulation of L-type VGCC (34). The regulation of {alpha}1C VGCC by PKC has been studied extensively. Subsequently, the NH2-terminal domain of {alpha}1C has been shown to be the main target for PKC in this modulation (21, 30). Interestingly, the characterization of the {alpha}1D VGCC modulation by PKC has not been assessed.

Eleven isozymes of PKC have been identified and divided into three groups based on their sequence homology and biochemical properties (8, 13, 23). These include the conventional PKCs ({alpha}, betaI, betaII, and {gamma}), regulated by Ca2+ and phorbol esters, the novel PKCs ({epsilon}, {delta}, {theta}, and {eta}), regulated by phorbol esters but not by Ca2+, and the atypical PKCs ({zeta} and {iota}/{lambda}), insensitive to both Ca2+ and phorbol esters. Various PKC isozymes are shown to have different intracellular distribution, and therefore each isozyme is suggested to exert different cellular function (6) and have different effects and mechanisms of action on ion channels (12, 35, 36).

The specific role of each PKC isozyme on L-type Ca2+ currents (ICaL) recorded from native myocytes or transfected cells has been made possible by the use of PKC modulator peptides that can inhibit individual endogenous PKC isozymes (31). We report for the first time that {alpha}1D VGCC is specifically regulated by betaII- and {epsilon}PKC isozymes. Molecular analysis of PKC-mediated regulation unraveled the implication of a serine residue located at position 81 of the {alpha}1D NH2-terminal domain as an important site for this regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasmids and site-directed mutagenesis. Rat pCMV6b/{alpha}1D plasmid was kindly provided by Dr. Susumu Seino (Kobe University, Kobe, Japan). Putative PKC phosphorylation sites with prediction score above the threshold were identified using NetPhos (http://www.cbs.dtu.dk/services/NetPhos) (3, 17). Site-directed mutagenesis was performed on pCMV6b/{alpha}1D using QuickChange TM site-directed mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer's instructions. Serine residues were replaced by alanine in all substitution mutants. The following mutations were introduced by mutagenic primers: S81A, S91A, S121A, and S98A/S100A to the pCMV6b/{alpha}1D (Fig. 1). Two NH2-terminal truncations were also constructed: {alpha}1D/{Delta}N(2–59) and {alpha}1D/{Delta}N(2–123). To delete amino acids 2 to 59 and 2 to 123, we introduced restriction enzyme XbaI followed by the Kozak sequence and ATG starting codon, using site-directed mutagenesis in pGFP37 constructs at the following positions: 177tctagaCCACCATG178 for {Delta}N(2–59) and 366tctagaCCACCATG367 for {Delta}N(2–123). Fragment XbaI was then removed. The presence of mutations was confirmed using the automatic sequencing at Laval University sequencing facility. Constructs were purified using Qiagen columns (Qiagen, Chatsworth, CA).


Figure 1
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  		Fig. 1. Amino acid sequence of the NH2-terminal domain from Cav1.3 channel and mutants. The drawing at top represents the secondary structure of Cav1.3 channels. Each of the 4 homologous domains consists of 6 membrane-spanning segments (S1–S6). The table at bottom shows the amino acid sequences for the Cav1.3 wild type (WT) and the mutants reported in this study. The shaded bars show the putative phosphorylation sites with the sequence number indicated above. Underlined bold type indicates the amino acid substitutions for the different mutants.

 
Transfection of tsA201 cell line. The mammalian cell line tsA201 is derived from human embryonic kidney HEK-293 cells by stable transfection with SV40 large-T antigen. Cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum, L-glutamine (2 mM), penicillin G (100 U/ml), and streptomycin (10 mg/ml; GIBCO BRL Life Technologies). Cells were incubated in a 5% CO2 humidified atmosphere. The tsA201 cells were transfected using the Ca2+ phosphate method with the following modification: to identify transfected cells, 7 µg of EBO/CD8 plasmid was cotransfected with 7 µg of each of {alpha}1D, beta, and {alpha}2/{delta} cDNAs. Transfected cells that bind beads generally also express Ca2+ channels. For patch-clamp experiments, cells 2–3 days posttransfection were incubated for 2 min in medium containing anti-CD8-a coated beads (M-450 CD8-a; Dynabeads) (14). The unattached beads were removed by washing with extracellular solution. Beads were prepared according to the manufacturer's instructions (Dynal Biotech, Brown Beer, WI). Cells expressing CD8-a, and therefore binding beads, were distinguished from nontransfected cells by light microscopy.

Solution, drugs, and peptides. For whole cell recordings, the pipette solution contained (in mM) 135 CsCl, 4 MgCl2, 4 ATP, 10 HEPES, 10 EGTA, and 1 EDTA, adjusted to pH 7.2 with tetraethylammonium hydroxide (TEAOH). The bath solution contained (in mM) 135 choline chloride, 1 MgCl2, 2 CaCl2, and 10 HEPES, adjusted to pH 7.2 with TEAOH. PKC isozyme peptides were added in the pipette solution as previously described (12, 35, 36). PKC isozyme-specific inhibitor and control peptides used in this study include {alpha}C2-4 ({alpha}PKC), betaIV5-3 (betaIPKC), betaIIV5-3 (betaIIPKC), {epsilon}V1-2 ({epsilon}PKC), {delta}V1-7 ({delta}PKC), scrambled {epsilon}V1-2, and pentalysine. The sequence and properties of PKC isozyme peptides were reported previously (12, 35, 36). Two additional peptides (peptide I and peptide II) corresponding to the NH2 terminus of {alpha}1D were also used in this study for both patch-clamp and in vitro phosphorylation studies. Peptide I corresponds to the NH2 terminus of {alpha}1D from amino acid 69 to 104 with all serine residues converted to alanines except for serine 81: N'-MATAAPPPVGALSQRKRQQYAKAKKQGNAANARPA-C'. For peptide II, all serine including the serine 81 were substituted by alanines: N'-MATAAPPPVGALAQRKRQQYAKAKKQGNAANARPA-C'.All peptides had >90% purity and were synthesized by Genemed Synthesis (South San Francisco, CA). PMA, 4{alpha}-phorbol 12,13-didecanoate (4{alpha}PDD), and GF 109203X were obtained from Sigma-Aldrich (St. Louis, MO).

Kinase assay. betaIIPKC and {epsilon}PKC kinase assay kits were purchased from Upstate Cell Signaling Solutions. For PKC kinase reactions, {alpha}1D NH2 terminus-derived peptides I and II (1.25 µg) were incubated withbetaIIPKC or {epsilon}PKC kinases. PKC kinase assays were performed in 25 µl of phosphorylation buffer containing (in mM) 200 HEPES, 1 CaCl2 (for betaIIPKC), 2.5 PKC lipid activator, pH 7.4, 0.3% Triton X-100, and 8.5 µCi {gamma}-[32P]ATP. Reactions were incubated for 30 min at 30°C, separated on 20% SDS-polyacrylamide gel, extensively washed, stained with Coomassie blue, fixed, and dried. [{gamma}-32P]ATP was detected by autoradiography.

Electrophysiology. Ca2+ currents were recorded in whole cell configuration of the patch-clamp technique (9). Data were digitized at 5 kHz with an analog-to-digital converter (Digidata 1200 Axon Instruments, Axon Instrument CA). The recordings were filtered with a low-pass corner frequency of 2 kHz. Borosilicate glass electrodes (outer diameter 1.5 mm) with resistances of 0.8–1.0 M{Omega} when filled were connected to a patch-clamp amplifier (Axopatch 200B amplifier). A voltage error of 7 mV, attributable to liquid junction potential, was corrected. Data were analyzed using pCLAMP version 9.0 (Axon Instruments).

Statistical analysis. Data were expressed as means ± SE. Percent inhibition was calculated as the difference of the current amplitude and the intervention over the control value. When indicated, paired or unpaired t-test was performed. Differences were deemed significant at a P value < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PMA inhibits {alpha}1D Ca2+ current. Representative inward {alpha}1D ICaL traces recorded before and after application of the general PKC activator PMA (10 nM) are shown in Fig. 2, A and B, respectively. PMA consistently inhibited {alpha}1D ICaL, and a steady-state inhibition was reached in all cells studied. For easy comparison, reduction in ICaL density (pA/pF) is represented as a percentage value throughout the text. Averaged current-voltage relations show that PMA reduced {alpha}1D ICaL density by 50.5 ± 5.0% (n = 9, P < 0.05; Fig. 2C) without significantly affecting steady-state activation [control: –25.0 ± 1.8 mV, n = 7 vs. PMA: –23.8 ± 2.2 mV, P = not significant (NS), n = 12; Fig. 2D] and inactivation curves (control: –35.4 ± 1.6 mV, n = 9 vs. PMA: –32.9 ± 1.4 mV, P = NS, n = 10; Fig. 2D). Slope factors were –5.7 ± 0.3 for control (n = 7, P =NS) compared with –5.5 ± 0.4 for PMA (n = 12) for activation and 7.6 ± 0.8 for control (n = 9, P = NS) compared with 7.0 ± 0.8 for PMA (n = 10) for inactivation. The time course for PMA-induced inhibition of {alpha}1D ICaL elicited at –10 mV is shown in Fig. 2E. For easy comparison, {alpha}1D ICaL are normalized to maximal amplitudes. To exclude the possibility that the current decay observed with PMA is due to a rundown, we carried out a set of control experiments. The time course of {alpha}1D ICaL recorded in the absence of PKC modulators shows no significant current reduction (n = 3; Fig. 3A). To confirm the specificity of PMA on {alpha}1D ICaL, we compared its effect to that of an inactive phorbol ester analog, 4{alpha}PDD, which does not activate PKC. Superfusion of tsA201 cells with 10 nM 4{alpha}PDD did not significantly affect {alpha}1D ICaL (reduction of 4.0 ± 1.7%, n = 3, P = NS; Fig. 3B), indicating that the observed PMA effect mentioned above is mediated through PKC activation. To further substantiate the role of PKC in the PMA inhibitory effect, we tested the ability of a general PKC inhibitor (GF 109203X) to antagonize the effect of PMA. Preincubation of {alpha}1D-expressing cells with GF 109203X (15 µM) for 10–15 min before superfusion with PMA resulted in only 7.0 ± 2.0% inhibition (n = 3, P = NS) of {alpha}1D ICaL after PMA application (Fig. 3C). In these experiments, GF 109203X (15 µM) alone did not affect {alpha}1D ICaL amplitude (data not shown). Because conventional PKC (cPKC) isozymes depend on Ca2+ for their activity (25), PMA effect was also assessed using a lower concentration of the Ca2+ chelator EGTA (0.1 mM) in the pipette (Fig. 3D). The results show that the PMA-induced inhibitory effect on ICaL was not significantly affected (42.1 ± 7%, n = 5, P = NS).


Figure 2
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  		Fig. 2. {alpha}1D L-type Ca2+ currents (ICaL) recorded from tsA201 cells. A: whole cell {alpha}1D ICaL traces recorded from tsA201 cells. Currents were recorded from a holding potential of –90 mV to test potentials from –80 to +50 mV for 250 ms in 10-mV increments using 2 mM Ca2+ as a charge carrier. B: whole cell {alpha}1D ICaL traces recorded after superfusion with the general PKC activator PMA (10 nM). C: current-voltage relationship for {alpha}1D ICaL (n = 9) before ({blacksquare}) and after PMA (bullet). D: steady-state activation and inactivation curves. Solid lines represent fits to the Boltzmann function finf(V) = 1/{1 + exp[(V1/2 – Vm)/k]} for steady-state activation and dinf(V) = 1/{1 + exp[(Vm – V1/2)/k]} for steady-state inactivation, where Vm is membrane voltage, V1/2 is the half-maximum inactivation potential, k is the slope factor, and dinf is the steady-state inactivation parameter. V1/2 values before and after PMA (10 nM) are, respectively, –25.0 ± 1.8 (n = 7,{blacktriangledown}) and –23.8 ± 2.2 mV (n = 12, {blacktriangleup}) for activation and –35.4 ± 1.6 (n = 9, bullet) and –32.9 ± 1.4 mV (n = 10, {blacksquare}) for inactivation. E: effect of PMA (10 nM) on {alpha}1D ICaL time course. Peak currents are plotted against time before and during superfusion of PMA. Inset illustrates {alpha}1D ICaL traces recorded at time points indicated by arrows a and b.

 

Figure 3
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  		Fig. 3. Time course of {alpha}1D ICaL recordings from tsA201 cells using 2 mM Ca2+ as a charge carrier. Currents were elicited at a test potential of –10 mV pulse protocol. A rundown test time course of {alpha}1D ICaL (A), the effect of 4{alpha}-phorbol 12,13-didecanoate (4{alpha}PDD; 10 nM) (B), and the effect of PMA (C) are shown in the presence of the general PKC inhibitor GF 109203X (15 µM). D: time course showing {alpha}1D ICaL inhibition following PMA (10 nM) superfusion when low EGTA (0.1 mM) concentration was used in the patch pipette. Peak currents are plotted against time before and during superfusion. Insets illustrate {alpha}1D ICaL traces recorded at time points indicated by arrows a and b.

 
PMA inhibition of {alpha}1D ICaL is mediated through serine 81 in NH2 terminus. Previous studies have shown that the NH2-terminal domain of {alpha}1C channel is critical for the PKC-mediated modulation (21, 30). In the present study, 11 putative PKC phosphorylation sites were predicted throughout {alpha}1D NH2-terminal domain as described in MATERIALS AND METHODS. To assess the role of these potential sites, deletion [{Delta}N(2–59) and {Delta}N(2–123)] and point mutation (S81A, S91A, S98A/S100A, and S121A) constructs were synthesized and studied in tsA201 cells (Fig. 1).

No current could be detected from {alpha}1D/{Delta}N(2–123) construct (data not shown). {alpha}1D/{Delta}N(2–59) deletant channel yielded a macroscopic {alpha}1D ICaL. This deletion did not prevent inhibition of {alpha}1D ICaL following superfusion with PMA (n = 10; Fig. 4A). This led us to rule out the existence of a functional PKC target within the segment of amino acid 2 to 59. We next investigated the role of five key potential phosphorylation residues in the remaining NH2-terminal portion. To this end, four {alpha}1D VGCC mutations were tested: one double substitution (S98A/S100A) and three single mutations (S81A, S91A, and S121A). The PMA effect on {alpha}1D ICaL was not significantly altered in {alpha}1D/S98A/S100A, {alpha}1D/S91A, and {alpha}1D/S121A constructs compared with the wild type (WT). The average percentages of {alpha}1D ICaL inhibition by PMA are 57.6 ± 4.8% (n = 5), 52.3 ± 5.1% (n = 5), and 52.6 ± 5.9% (n = 7) vs. 50.5 ± 5.0% (n = 9, P = NS), respectively (Fig. 4, B–D). Interestingly, PMA-induced {alpha}1D ICaL inhibition was antagonized in the presence of S81A mutation (15.0 ± 8.2%, n = 9, P = NS), suggesting a critical role for this particular site in PKC-mediated modulation of the {alpha}1D subunit (Fig. 4E). Averaged data from all mutations are summarized in Fig. 4F. It is noteworthy that the presence of mutations altered neither the kinetics nor the gating properties of the {alpha}1D VGCC.


Figure 4
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  		Fig. 4. Effects of PMA (10 nM) on {alpha}1D mutant channels. Time courses of {alpha}1D ICaL were recorded from a tsA201 cell expressing {alpha}1D/{Delta}N(2–59) (A), {alpha}1D/S98A/S100A (B), {alpha}1D/S91A (C), {alpha}1D/S121A (D), and {alpha}1D/S81A mutants (E). Insets illustrate current traces recorded at time points indicated by arrows a and b. F: histogram showing percentage of inhibition of {alpha}1D ICaL in the presence of PMA (10 nM) recorded from different mutants. Currents amplitudes are normalized against the control (before PMA). Data are means ± SE. *P < 0.05, statistically significant difference compared with {alpha}1D/WT after superfusion of PMA (10 nM).

 
To substantiate the role of serine 81 in the PMA inhibitory effect, we tested the ability of a peptide of 35 amino acids (peptide I) that corresponded to the NH2-terminal region comprising residues 69–104 and where all serine residues, except for serine 81, were converted to alanines (see MATERIALS AND METHODS) to antagonize the effect of PMA on {alpha}1D channels. Cells were dialyzed with 10 µM peptide-I for at least 5 min to allow for the peptide to access the cytoplasm. PMA perfusion of cells initially dialyzed with peptide I resulted in only 30.7 ± 5.8% inhibition (n = 7, P < 0.05) of basal {alpha}1D ICaL compared with control (no peptides were dialyzed into cells) (Fig. 5A). However, when similar experiments were repeated using a control peptide (peptide II) in which all the serine residues were replaced with alanines, the effect of PMA on {alpha}1D ICaL was not prevented by the presence of peptide II (10 µM). PMA inhibited {alpha}1D ICaL by 53.3 ± 7.2% (n = 8, P = NS) in cells dialyzed with peptide II.


Figure 5
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  		Fig. 5. Histograms showing percentage of inhibition of {alpha}1D ICaL in the presence of PMA (10 nM) recorded from control cells vs. cells dialyzed with peptides I and II (A) and cells expressing WT vs. S81A and S81D {alpha}1D mutant channels (B). Current amplitudes are normalized against {alpha}1D ICaL before superfusion with PMA. Data are means ± SE. *P < 0.05, statistically significant difference compared with {alpha}1D ICaL inhibition by PMA in control cells.

 
To determine the mechanism by which serine 81 is involved in {alpha}1D modulation by PKC, we substituted this neutral residue with the negatively charged aspartate, to mimic the negative charge introduced during naturally occurring phosphorylation. In the absence of PMA, conversion of serine (neutral amino acid) to aspartate (negative amino acid) at position 81 resulted in a significant reduction of {alpha}1D ICaL by 42.2 ± 7.8% (n = 20, P < 0.05 compared with WT). In the presence of a negative charge at position 81, PMA did not have significant inhibitory effect on {alpha}1D ICaL (6.23 ± 4.5%, n = 9) (Fig. 5B). These data suggest that gain of negative charge at position 81 in the NH2-terminal domain could be the molecular mechanism by which {alpha}1D subunit is modulated by PKC.

PMA inhibition of {alpha}1D ICaL is mediated through betaII- and {epsilon}PKC isozymes. To dissect the role of individual PKC isozymes in the regulation of {alpha}1D VGCC, we tested the ability of PKC isozyme-specific inhibitor peptides (31) to antagonize the effect of PMA. Six PKC isozyme-specific inhibitor peptides were used: {alpha}C2-4, betaIV5-3, betaIIV5-3, {delta}V1-2, {epsilon}V1-2, and {eta}V1-2 targeting {alpha}-, betaI-, betaII-, {delta}-, {epsilon}-, and {eta}PKCs, respectively. For each set of experiments, cells were dialyzed with a different inhibitor (0.1 µM) for at least 5 min to allow for peptides to access the cytoplasm. These peptides alone did not have any effect on {alpha}1D ICaL. Subsequently, the PMA inhibitory effect on {alpha}1D ICaL was assessed in the presence of each of these six inhibitor peptides, respectively. PMA superfusion of cells initially dialyzed with {alpha}C2-4, betaIV5-3, {delta}V1-2, or {eta}V1-2 (0.1 µM) did not prevent {alpha}1D ICaL inhibition. Average percentages of inhibition were 43.0 ± 4.0% for {alpha}C2-4 (n = 9, P = NS), 50.0 ± 6.0% for betaIV5-3 (n = 4, P = NS), 47.6 ± 5.0% for {delta}V1-2 (n = 9, NS), and 48.3 ± 5% for {eta}V1-2 compared with control (where cells were not dialyzed with any peptide, 50.5 ± 5.0%, n = 7). In contrast, PMA superfusion of cells dialyzed with betaIIV5-3 (0.1 µM) resulted in 22.0 ± 7.0% inhibition (n = 7, P < 0.05) of {alpha}1D ICaL (Fig. 6A). Similarly, in cells dialyzed with {epsilon}V1-2 peptide inhibitor, PMA resulted in only 11.0 ± 5.0% inhibition (n = 5, P < 0.05) of {alpha}1D ICaL (Fig. 6B). Next, we tested whether betaIIV5-3 and {epsilon}V1-2 peptides have an additive antagonizing effect of {alpha}1D ICaL inhibition by PMA. Simultaneous application of both betaIIV5-3 (0.1 µM) and {epsilon}V1-2 (0.1 µM) in pipette solution completely abolished the inhibitory effect of PMA (0.5 ± 0.1%, n = 5, P < 0.05; Fig. 6C). In vitro phosphorylation reactions using NH2 terminus-derived peptides II and I showed that only peptide I, containing serine 81, was phosphorylated by betaIIPKC and {epsilon}PKC (Fig. 6D). These results indicate that serine 81 is required for {alpha}1D subunit phosphorylation by betaIIPKC and {epsilon}PKC.


Figure 6
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  		Fig. 6. Effect of PMA on {alpha}1D ICaL in the presence of PKC isozyme-specific inhibitor peptides. Time courses of {alpha}1D ICaL were recorded from tsA201 cells dialyzed with peptide inhibitors for betaIIPKC (betaIIV5-3, 0.1 µM) (A), {epsilon}PKC ({epsilon}V1-2, 0.1 µM) (B), and betaIIPKC + {epsilon}PKC (betaIIV5-3, 0.1 µM + {epsilon}V1-2, 0.1 µM) (C). Insets illustrate {alpha}1D ICaL traces recorded at time points indicated by arrows a and b. D: in vitro phosphorylation of {alpha}1D NH2 terminus-derived peptides I and II in the presence of betaIIPKC and {epsilon}PKCs.

 
Additional patch-clamp experiments were performed in the presence of pentalysine or a scrambled {epsilon}V1-2 as negative controls (Fig. 7). The inhibitory effect of PMA on {alpha}1D ICaL was not prevented in the presence of scrambled {epsilon}V1-2 (0.1 µM; 47.0 ± 2.8, n = 5, P < 0.05) or pentalysine (0.1 µM; 46.5 ± 3.9, n = 5, P < 0.05). These findings demonstrate that betaIIPKC and {epsilon}PKC isozymes are selectively involved in the modulation of {alpha}1D VGCC expressed in tsA201 cells. Data from different peptides are summarized in the histogram of Fig. 7.


Figure 7
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  		Fig. 7. Summary histogram showing the percentage of {alpha}1D ICaL changes in response to PMA (10 nM) in the presence of various PKC isozyme-specific peptides. GF-X, GF 109203X.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study we provide novel functional and biochemical evidence for {alpha}1D VGCC regulation by PKC through a serine residue located at position 81 in the NH2-terminal domain of the {alpha}1D subunit. Using the HEK-293-derived tsA201 cells known to endogenously express most of phorbol ester-sensitive PKC isozymes, including {alpha}, betaI, betaII, {delta}, and {epsilon} (18), we have shown that {alpha}1D ICaL is inhibited by 50% following superfusion with the general PKC activator PMA. This inhibitory effect is specifically attributed to the activation of PKC given the fact that similar effect could not be reproduced using the biologically inactive phorbol ester analog 4{alpha}PDD, which does not activate PKC. In addition, the {alpha}1D ICaL inhibition by PMA was prevented by a known general PKC inhibitor, GF 109203X. A time-dependent ICaL rundown effect (2) has been ruled out, because no variations in ICaL amplitudes were observed in control experiments. Together, these data confirm that {alpha}1D VGCC is regulated by PKC.

Phosphorylation of NH2-terminal serines by PKC. One of the main novel observations in the present work is that a single serine mutation (S81A) at the NH2-terminal domain of {alpha}1D subunit readily confers a dramatic lack of PMA effect on {alpha}1D ICaL. To determine whether the NH2-terminal domain of {alpha}1D subunit is involved in channel phosphorylation by PKC, deletions [{Delta}N(2–59) and {Delta}N(2–123)] and individual point mutations of putative phosphorylation sites in which serines were mutated to alanine residues (S81A, S91A, S121A, and S98A/S100A) were tested in the presence of PMA. Interestingly, only the substitution of serine at position 81 by an alanine (S81A) prevented most of the PMA effect. Furthermore, intracellular application of peptide I, corresponding to the {alpha}1D NH2-terminal segment that contains serine 81, significantly reduced the inhibitory effect of PMA on {alpha}1D/WT channels, consistent with data obtained in the presence of S81A mutation. This finding suggests that peptide I directly competes with the corresponding NH2-terminal sequence on {alpha}1D/WT subunits expressed in tsA201 cells. Furthermore, introducing a negatively charged residue at this site (S81D) resulted in smaller {alpha}1D ICaL amplitudes compared with WT, therefore mimicking the inhibitory effect of PKC activation on intact {alpha}1D channel. These data support the conclusion that serine 81 is a crucial site for {alpha}1D subunit phosphorylation by PKC and suggest that the gain of a negative charge at position 81 could be the molecular mechanism by which {alpha}1D subunit is modulated by PKC.

Because expression of {alpha}1D subunit alone in tsA201 cells yielded no functional current in the present study and a previous study (28), {alpha}1D subunit was coexpressed with the beta and {alpha}2/{delta} accessory subunits in tsA201 cells. In this regard, we do not completely rule out the potential phosphorylation of these accessory subunits (essentially beta subunit) by PKC. However, the resulting effect must be minimal, because S81 mutation of the pore-forming {alpha}1D subunit antagonized the majority of the PMA effect on {alpha}1D ICaL.

PKC isozyme-specific modulation of {alpha}1D Ca2+ channels. We dissected the role of individual PKC isozymes in the regulation of {alpha}1D VGCC using the established PKC isozyme-specific inhibitor peptides (31). Two PKC isozymes were found to be involved in {alpha}1D ICaL inhibition by PMA: betaIIPKC and {epsilon}PKC. Whereas inhibition of {epsilon}PKC antagonized ~78% of the PMA effect (inhibition of {alpha}1D ICaL was reduced from 50.5 to 11%), the inhibition of betaIIPKC reduced it by 56% (inhibition of {alpha}1D ICaL was reduced from 50.5 to 22%). A combination of both betaIIPKC and {epsilon}PKC inhibitors into the pipette prevented almost 100% of the inhibitory effect of PMA. None of the other PKC isozyme-specific inhibitor peptides ({alpha}-, betaI-, {delta}-, and {eta}PKCs) significantly altered the PMA-induced inhibition of {alpha}1D ICaL. These results indicate that betaIIPKC and {epsilon}PKC mediate the {alpha}1D VGCC inhibition by PMA. Because members of cPKC ({alpha}, betaI, betaII, and {gamma}) are known to depend on Ca2+ in their activity (25), the effects of the corresponding inhibitors were studied at 10 and 0.1 mM EGTA (Figs. 2E and 3D) in the pipette solution. Similar effects were observed at either concentration. Indeed, under our experimental conditions, cPKC activation in the presence of EGTA can be attributed to the extracellular Ca2+ that we used as charge carrier and to the fact that phorbol esters are known to increase the affinity of cPKC for Ca2+ (24).

Study limitations. Because we used the whole cell configuration of the patch-clamp experiments, we cannot rule out the possibility that the quantitative aspects of the response to PMA in the presence of peptide PKC isozyme modulators might differ in a nondialyzed cell system. Introduction of mutation(s) to a channel may interfere with channel protein trafficking. In this study, we did not assess all the factors that may affect expression of a mutated channel, including trafficking. Therefore, we cannot completely rule out potential effects these factors may have on the expression of the mutated channel in this study.

Pathophysiological significance. In the cardiovascular system, voltage-gated L-type Ca2+ channels are essential for the generation of normal cardiac rhythm, for induction of rhythm propagation through the atrioventricular (AV) node, and for the contraction of the atrial and ventricular muscle. In diseased myocardium, L-type Ca2+ channels can contribute to abnormal impulse generation and cardiac arrhythmias (11). The {alpha}1D Ca2+ channel is generally termed neuroendocrine because of its broad distribution in the brain (19) and insulin-secreting beta cells of the pancreas (29). The function of {alpha}1D Ca2+ channel in the heart had long been ignored until recent studies showed that {alpha}1D Ca2+ channel knockout mice unexpectedly exhibited intrinsic sinoatrial and AV node dysfunction (20, 26, 39). At the whole animal level, significant prolongation of the R-R and PR intervals in surface ECG were observed in {alpha}1D knockout mice (20, 26, 39). More recently, atrial arrhythmias, mainly atrial fibrillation, were induced in all {alpha}1D Ca2+ channel knockout mice, indicating a role of {alpha}1D ICaL in the sinoatrial tissue (38). Furthermore, we also recently showed that {alpha}1D ICaL recorded from tsA201 cells is modulated by protein kinase A (PKA) (20, 26–28, 39) and may play a role in the pathophysiology of autoimmune-associated congenital heart block (27). Altogether, the above recent data demonstrate that {alpha}1D Ca2+ channels play an important role in the heart, and thus the mechanisms of its regulation by kinases such as PKA and PKC are relevant. Because {alpha}1D ICaL in native cardiac myocytes cannot be distinguished pharmacologically from {alpha}1C ICaL because of the absence of selective modulators (20, 26–28, 39), the characterization of {alpha}1D Ca2+ channel modulation by PKA/PKC is limited to mammalian tsA201 cells.

In conclusion, the results establish that {alpha}1D VGCC is regulated by PKC. This regulation involves phosphorylation of {alpha}1D NH2-terminal domain. Specifically, we have shown that serine 81 represents a critical site for PKC-mediated regulation of {alpha}1D VGCC and pointed to betaIIPKC and {epsilon}PKC as the key isozyme players in this regulation. Understanding the molecular mechanism of {alpha}1D VGCC regulation through PKC provides novel insights in the development of new drugs that interfere with specific components of the channel protein and/or its regulatory pathways. The results also suggest that {epsilon}PKC and betaIIPKC isozymes may constitute suitable candidates for the development of targeted therapeutic for cardiac pathophysiology involving {alpha}1D Ca2+ channels, such as atrial fibrillation or autoimmune-associated congenital heart block.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work is supported by a Veterans Affairs Merit Grant and National Heart, Lung, and Blood Institute Grant R01 HL-077494 to M. Boutjdir, a fellowship grant from the Canadian Institutes of Health Research (CIHR) to G. Baroudi, and a Veterans Affairs Merit Review Entry Program Grant (to Y. Qu). This study also was supported by grants from the Heart and Stroke Foundation of Quebec and CIHR Grant MT-13181 to M. Chahine, who is an Edwards Senior Investigator (Joseph C. Edwards Foundation).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Boutjdir, Veterans Affairs New York Harbor Healthcare System, Research and Development (151), 800 Poly Place, Brooklyn, NY 11209 (e-mail: mohamed.boutjdir{at}med.va.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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