Am J Physiol Heart Circ Physiol 291: H1614-H1622, 2006;
doi:10.1152/ajpheart.00095.2006
0363-6135/06 $8.00
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
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ABSTRACT
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The Cav1.3 (
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
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
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
1D Ca2+ channel. The modulation of
1D Ca2+ channel by PKC was prevented by dialyzing cells with a 35-amino acid peptide mimicking the
1D NH2-terminal region comprising S81. In addition, the data revealed that only
II- and
PKC isozymes are implicated in this regulation. These novel findings have significant implications in the pathophysiology of
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
1S and
1F are mainly restricted to skeletal muscle and retinal neuron, respectively (1, 4, 16, 22), the
1C is expressed in the heart, vascular smooth muscles, and neurons (10, 32). More recently,
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,
1D VGCC knockout mice exhibited sinus bradycardia and atrioventricular block (20), and
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
1C VGCC by PKC has been studied extensively. Subsequently, the NH2-terminal domain of
1C has been shown to be the main target for PKC in this modulation (21, 30). Interestingly, the characterization of the
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 (
,
I,
II, and
), regulated by Ca2+ and phorbol esters, the novel PKCs (
,
,
, and
), regulated by phorbol esters but not by Ca2+, and the atypical PKCs (
and
/
), 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
1D VGCC is specifically regulated by
II- and
PKC isozymes. Molecular analysis of PKC-mediated regulation unraveled the implication of a serine residue located at position 81 of the
1D NH2-terminal domain as an important site for this regulation.
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MATERIALS AND METHODS
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Plasmids and site-directed mutagenesis.
Rat pCMV6b/
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/
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/
1D (Fig. 1). Two NH2-terminal truncations were also constructed:
1D/
N(259) and
1D/
N(2123). 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
N(259) and 366tctagaCCACCATG367 for
N(2123). 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).

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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 (S1S6). 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.
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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
1D,
, and
2/
cDNAs. Transfected cells that bind beads generally also express Ca2+ channels. For patch-clamp experiments, cells 23 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
C2-4 (
PKC),
IV5-3 (
IPKC),
IIV5-3 (
IIPKC),
V1-2 (
PKC),
V1-7 (
PKC), scrambled
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
1D were also used in this study for both patch-clamp and in vitro phosphorylation studies. Peptide I corresponds to the NH2 terminus of
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
-phorbol 12,13-didecanoate (4
PDD), and GF 109203X were obtained from Sigma-Aldrich (St. Louis, MO).
Kinase assay.
IIPKC and
PKC kinase assay kits were purchased from Upstate Cell Signaling Solutions. For PKC kinase reactions,
1D NH2 terminus-derived peptides I and II (1.25 µg) were incubated with
IIPKC or
PKC kinases. PKC kinase assays were performed in 25 µl of phosphorylation buffer containing (in mM) 200 HEPES, 1 CaCl2 (for
IIPKC), 2.5 PKC lipid activator, pH 7.4, 0.3% Triton X-100, and 8.5 µCi
-[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. [
-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 0.81.0 M
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.
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RESULTS
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PMA inhibits
1D Ca2+ current.
Representative inward
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
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
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
1D ICaL elicited at 10 mV is shown in Fig. 2E. For easy comparison,
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
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
1D ICaL, we compared its effect to that of an inactive phorbol ester analog, 4
PDD, which does not activate PKC. Superfusion of tsA201 cells with 10 nM 4
PDD did not significantly affect
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
1D-expressing cells with GF 109203X (15 µM) for 1015 min before superfusion with PMA resulted in only 7.0 ± 2.0% inhibition (n = 3, P = NS) of
1D ICaL after PMA application (Fig. 3C). In these experiments, GF 109203X (15 µM) alone did not affect
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).

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Fig. 2. 1D L-type Ca2+ currents (ICaL) recorded from tsA201 cells. A: whole cell 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 1D ICaL traces recorded after superfusion with the general PKC activator PMA (10 nM). C: current-voltage relationship for 1D ICaL (n = 9) before ( ) and after PMA ( ). 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, ) and 23.8 ± 2.2 mV (n = 12, ) for activation and 35.4 ± 1.6 (n = 9, ) and 32.9 ± 1.4 mV (n = 10, ) for inactivation. E: effect of PMA (10 nM) on 1D ICaL time course. Peak currents are plotted against time before and during superfusion of PMA. Inset illustrates 1D ICaL traces recorded at time points indicated by arrows a and b.
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PMA inhibition of
1D ICaL is mediated through serine 81 in NH2 terminus.
Previous studies have shown that the NH2-terminal domain of
1C channel is critical for the PKC-mediated modulation (21, 30). In the present study, 11 putative PKC phosphorylation sites were predicted throughout
1D NH2-terminal domain as described in MATERIALS AND METHODS. To assess the role of these potential sites, deletion [
N(259) and
N(2123)] 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
1D/
N(2123) construct (data not shown).
1D/
N(259) deletant channel yielded a macroscopic
1D ICaL. This deletion did not prevent inhibition of
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
1D VGCC mutations were tested: one double substitution (S98A/S100A) and three single mutations (S81A, S91A, and S121A). The PMA effect on
1D ICaL was not significantly altered in
1D/S98A/S100A,
1D/S91A, and
1D/S121A constructs compared with the wild type (WT). The average percentages of
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, BD). Interestingly, PMA-induced
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
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
1D VGCC.
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 69104 and where all serine residues, except for serine 81, were converted to alanines (see MATERIALS AND METHODS) to antagonize the effect of PMA on
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
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
1D ICaL was not prevented by the presence of peptide II (10 µM). PMA inhibited
1D ICaL by 53.3 ± 7.2% (n = 8, P = NS) in cells dialyzed with peptide II.
To determine the mechanism by which serine 81 is involved in
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
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
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
1D subunit is modulated by PKC.
PMA inhibition of
1D ICaL is mediated through
II- and
PKC isozymes.
To dissect the role of individual PKC isozymes in the regulation of
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:
C2-4,
IV5-3,
IIV5-3,
V1-2,
V1-2, and
V1-2 targeting
-,
I-,
II-,
-,
-, and
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
1D ICaL. Subsequently, the PMA inhibitory effect on
1D ICaL was assessed in the presence of each of these six inhibitor peptides, respectively. PMA superfusion of cells initially dialyzed with
C2-4,
IV5-3,
V1-2, or
V1-2 (0.1 µM) did not prevent
1D ICaL inhibition. Average percentages of inhibition were 43.0 ± 4.0% for
C2-4 (n = 9, P = NS), 50.0 ± 6.0% for
IV5-3 (n = 4, P = NS), 47.6 ± 5.0% for
V1-2 (n = 9, NS), and 48.3 ± 5% for
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
IIV5-3 (0.1 µM) resulted in 22.0 ± 7.0% inhibition (n = 7, P < 0.05) of
1D ICaL (Fig. 6A). Similarly, in cells dialyzed with
V1-2 peptide inhibitor, PMA resulted in only 11.0 ± 5.0% inhibition (n = 5, P < 0.05) of
1D ICaL (Fig. 6B). Next, we tested whether
IIV5-3 and
V1-2 peptides have an additive antagonizing effect of
1D ICaL inhibition by PMA. Simultaneous application of both
IIV5-3 (0.1 µM) and
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
IIPKC and
PKC (Fig. 6D). These results indicate that serine 81 is required for
1D subunit phosphorylation by
IIPKC and
PKC.
Additional patch-clamp experiments were performed in the presence of pentalysine or a scrambled
V1-2 as negative controls (Fig. 7). The inhibitory effect of PMA on
1D ICaL was not prevented in the presence of scrambled
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
IIPKC and
PKC isozymes are selectively involved in the modulation of
1D VGCC expressed in tsA201 cells. Data from different peptides are summarized in the histogram of Fig. 7.

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Fig. 7. Summary histogram showing the percentage of 1D ICaL changes in response to PMA (10 nM) in the presence of various PKC isozyme-specific peptides. GF-X, GF 109203X.
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DISCUSSION
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In the present study we provide novel functional and biochemical evidence for
1D VGCC regulation by PKC through a serine residue located at position 81 in the NH2-terminal domain of the
1D subunit. Using the HEK-293-derived tsA201 cells known to endogenously express most of phorbol ester-sensitive PKC isozymes, including
,
I,
II,
, and
(18), we have shown that
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
PDD, which does not activate PKC. In addition, the
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
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
1D subunit readily confers a dramatic lack of PMA effect on
1D ICaL. To determine whether the NH2-terminal domain of
1D subunit is involved in channel phosphorylation by PKC, deletions [
N(259) and
N(2123)] 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
1D NH2-terminal segment that contains serine 81, significantly reduced the inhibitory effect of PMA on
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
1D/WT subunits expressed in tsA201 cells. Furthermore, introducing a negatively charged residue at this site (S81D) resulted in smaller
1D ICaL amplitudes compared with WT, therefore mimicking the inhibitory effect of PKC activation on intact
1D channel. These data support the conclusion that serine 81 is a crucial site for
1D subunit phosphorylation by PKC and suggest that the gain of a negative charge at position 81 could be the molecular mechanism by which
1D subunit is modulated by PKC.
Because expression of
1D subunit alone in tsA201 cells yielded no functional current in the present study and a previous study (28),
1D subunit was coexpressed with the
and
2/
accessory subunits in tsA201 cells. In this regard, we do not completely rule out the potential phosphorylation of these accessory subunits (essentially
subunit) by PKC. However, the resulting effect must be minimal, because S81 mutation of the pore-forming
1D subunit antagonized the majority of the PMA effect on
1D ICaL.
PKC isozyme-specific modulation of
1D Ca2+ channels.
We dissected the role of individual PKC isozymes in the regulation of
1D VGCC using the established PKC isozyme-specific inhibitor peptides (31). Two PKC isozymes were found to be involved in
1D ICaL inhibition by PMA:
IIPKC and
PKC. Whereas inhibition of
PKC antagonized
78% of the PMA effect (inhibition of
1D ICaL was reduced from 50.5 to 11%), the inhibition of
IIPKC reduced it by 56% (inhibition of
1D ICaL was reduced from 50.5 to 22%). A combination of both
IIPKC and
PKC inhibitors into the pipette prevented almost 100% of the inhibitory effect of PMA. None of the other PKC isozyme-specific inhibitor peptides (
-,
I-,
-, and
PKCs) significantly altered the PMA-induced inhibition of
1D ICaL. These results indicate that
IIPKC and
PKC mediate the
1D VGCC inhibition by PMA. Because members of cPKC (
,
I,
II, and
) 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
1D Ca2+ channel is generally termed neuroendocrine because of its broad distribution in the brain (19) and insulin-secreting
cells of the pancreas (29). The function of
1D Ca2+ channel in the heart had long been ignored until recent studies showed that
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
1D knockout mice (20, 26, 39). More recently, atrial arrhythmias, mainly atrial fibrillation, were induced in all
1D Ca2+ channel knockout mice, indicating a role of
1D ICaL in the sinoatrial tissue (38). Furthermore, we also recently showed that
1D ICaL recorded from tsA201 cells is modulated by protein kinase A (PKA) (20, 2628, 39) and may play a role in the pathophysiology of autoimmune-associated congenital heart block (27). Altogether, the above recent data demonstrate that
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
1D ICaL in native cardiac myocytes cannot be distinguished pharmacologically from
1C ICaL because of the absence of selective modulators (20, 2628, 39), the characterization of
1D Ca2+ channel modulation by PKA/PKC is limited to mammalian tsA201 cells.
In conclusion, the results establish that
1D VGCC is regulated by PKC. This regulation involves phosphorylation of
1D NH2-terminal domain. Specifically, we have shown that serine 81 represents a critical site for PKC-mediated regulation of
1D VGCC and pointed to
IIPKC and
PKC as the key isozyme players in this regulation. Understanding the molecular mechanism of
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
PKC and
IIPKC isozymes may constitute suitable candidates for the development of targeted therapeutic for cardiac pathophysiology involving
1D Ca2+ channels, such as atrial fibrillation or autoimmune-associated congenital heart block.
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GRANTS
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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).
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FOOTNOTES
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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.
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