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Am J Physiol Heart Circ Physiol 288: H2123-H2130, 2005. First published December 22, 2004; doi:10.1152/ajpheart.01023.2004
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Localization and modulation of {alpha}1D (Cav1.3) L-type Ca channel by protein kinase A

Yongxia Qu, Ghayath Baroudi, Yuankun Yue, Nabil El-Sherif, and Mohamed Boutjdir

Veterans Affairs New York Harbor Healthcare System, State University of New York Downstate Medical Center, Brooklyn and New York School of Medicine, New York, New York

Submitted 7 October 2004 ; accepted in final form 19 December 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{alpha}1D L-type Ca channel was assumed to be of neuroendocrine origin only; however, {alpha}1D L-type Ca channel knockout mice exhibit sinus bradycardia and atrioventricular block, indicating a distinct role of {alpha}1D in the heart. The presence and distribution of {alpha}1D Ca channel in the heart and its regulation by protein kinase A (PKA) are just emerging. Our objective was to examine the localization of {alpha}1D L-type Ca channel in rabbit and rat hearts and its modulation by PKA. Here, we show the exclusive presence of {alpha}1D Ca channel transcript in the sinoatrial node, atrioventricular node, and atria but not in the ventricle by RT-PCR and the expression of {alpha}1D Ca channel protein in atrial myocytes' sarcolemma by indirect immunostaining and Western blot. There is no significant difference in the expression level of {alpha}1D Ca channel in the left versus right atrium. Superfusion of membrane-permeable 8-bromo-cAMP resulted in a significant increase of the peak current density of {alpha}1D Ca current expressed in tsA201 cells. This increase was inhibited by the PKA inhibitor (PKI). Application of 8-bromo-cAMP also readily phosphorylated the {alpha}1D Ca channel protein. The results are first to demonstrate that PKA phosphorylation of L-type Ca channel {alpha}1D-subunit resulted in an increase of the {alpha}1D Ca channel activity. Together with the observation that {alpha}1D Ca channel is exclusively present in the sinoatrial node and atria, the findings suggest that {alpha}1D Ca channel plays a unique role in the sinoatrial tissue and is a target for sympathetic control of heart rhythm.

phosphorylation; protein kinase A; sinoatrial node

IN THE CARDIOVASCULAR SYSTEM, voltage-gated L-type Ca channels are essential for the generation of normal cardiac rhythm, for the induction of rhythm propagation through the atrioventricular (AV) node, and for the contraction of the atrial and ventricular muscle (31). Cardiac L-type Ca channels are heterologomeric complexes of up to four subunits: {alpha}1-, {beta}-, and {alpha}2/{delta} subunits. The {alpha}1-subunit contains the voltage sensor, the selectivity filter, the ion-conducting pore, and the binding sites for all known Ca channel blockers. Four genes encode L-type Ca channel {alpha}1-subunits in mammals (alpha S, C, D, and F) (7). Alpha S ({alpha}1S) and alpha F ({alpha}1F) expression is restricted to skeletal muscle and the retina. Alpha C ({alpha}1C) represents the most abundant isoform in the cardiovascular system, whereas alpha D ({alpha}1D) is mainly expressed in neurons and neuroendocrine cells.

It has therefore long been assumed that the contribution of L-type Ca current (ICa-L) to physiology and/or pathophysiology of the heart and the therapeutic effects of Ca channel blockers are mainly mediated through {alpha}1C Ca channel. This assumption has been challenged by recent reports indicating a role of {alpha}1D Ca channel in the pacemaker activity of the sinoatrial (SA) node. Takimoto et al. (37) reported that {alpha}1D Ca channel mRNA is expressed in the rat atrium but not in the ventricles. This is subsequently confirmed by Mangoni et al. (19) who reported a similar observation in mouse and human hearts. {alpha}1D differs from {alpha}1C Ca channel in that it is activated at more negative membrane potential (2, 39). This property allows {alpha}1D Ca channel to play a more important role in diastolic depolarization. Indeed, {alpha}1D Ca channel knockout mice exhibited significant sinus bradycardia and AV block (19, 27, 43). The density of ICa-L was reduced by 79% in {alpha}1D Ca channel knockout mice compared with wild-type mice (19), indicating the significant contribution of {alpha}1D Ca channel to the total ICa-L in the SA node and challenge the view of the minor contribution of L-type Ca channel into diastolic depolarization of the SA node. Nevertheless and despite the elegant work on {alpha}1D Ca channel in the SA node, the information on {alpha}1D Ca channel in the atria is still lacking.

The SA tissue is heavily innervated by the sympathetic nervous system (SNS) (30). The stimulation of the SNS in response to exercise or stress insults results in a rapid and dramatic increase in heart rate. SNS control of SA node activity is mediated by the activation of {beta}-adrenergic receptors that regulate the activity of select ion-channel proteins via cAMP-dependent PKA (36). cAMP-PKA is also the converging point of regulation of the Ca channel by many neurotransmitters and hormone receptors, including the serotoninergic 5-HT4 receptor and {beta}1- and {beta}2-adrenoreceptors, which are identified in human atrial cells (8, 14, 20, 25, 35, 40). Given the presence of {alpha}1D in both the SA node and atria, it is important to understand whether and how PKA modulates {alpha}1D ICa-L. It has been well established that PKA stimulation increases {alpha}1C ICa-L (22). However, whether or not {alpha}1D ICa-L is modulated by cAMP-PKA in a similar way has not yet been reported.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and antibodies. 8-Bromo-cAMP, cell permeable cAMP-dependent PKA inhibitor (PKI, myristoylated trifluoroacetate salt), anti-{alpha}1D Ca channel antibody, and all other chemicals used in this study were purchased from Sigma. The anti-{alpha}1D Ca channel antibody is developed in the rabbit by using a synthetic peptide corresponding to amino acids 809–825 of the {alpha}1D-subunit of rat L-type Ca channel.

Rabbit SA/AV node isolation. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the Veterans Affairs New York Harbor Healthcare System. New Zealand White rabbits were anesthetized with intravenous injection of pentobarbital sodium (40 mg/kg). The heart was rapidly excised and immersed in a normal Tyrode solution containing (in mmol/l): 140 NaCl, 5.4 KCl, 1.0 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, and 5 HEPES (pH = 7.4). Rabbit SA and AV node tissue was prepared according to Denyer and Brown (6) and Hancox et al. (10), respectively. The SA node and AV node tissue were snap frozen in liquid nitrogen.

RNA preparation. Total cellular RNA was isolated from adult rabbit SA node, AV node, right atrium (RA), and right ventricle (RV) using RNAzol B kit (TEL-TEST) as previously described (28, 29). Residual genomic DNA was digested with RQ DNase (5 U, Promega) for 15 min at 37°C, and the reaction was terminated by extraction with phenol. RNA was quantified by spectrophotometry at 260 nm, and the ratio of absorbance at 260 nm to that of 280 nm was >1.8 for all samples. Degradation of RNA samples was monitored by the observation of appropriate 28S to 18S ribosomal RNA ratios as determined by ethidium bromide staining of the agarose gels.

RT-PCR. Reverse transcription was carried out using Maxiscript kit from Ambion as previously described (29, 36). The resulting cDNA was amplified by PCR. The sense primer is 5'-TTAGTGACGCCTGGAACACG-3', and the antisense primer is 5'-CCTGTATCAGGAAAGTGG-3'. The expected PCR amplification size is 1047 bp. PCR was carried out in a volume of 50 µl containing 2 units of Taq DNA polymerase (Ambion) in the supplied buffer supplemented with 200 µM of each dNTP and 0.4 µM of each primer. The following cycles were used: 94°C for 5 min (1 cycle), 94°C for 60 s, 55°C for 60 s, 72°C for 1 min (30 cycles), and 72°C for 10 min (1 cycle). Final PCR products were evaluated on ethidium bromide-stained 1% agarose gel. The sequencing of the PCR products was performed by Genemed.

Isolation of cardiac myocytes. Cardiac myocytes were obtained from Langendorff perfused 6-mo-old rat hearts as previously described (4, 42). Hearts were perfused at 37°C with a HEPES-buffered solution containing (in mmol/l): 117 NaCl, 5.7 KCl, 4.4 NaHCO3, 1.5 NaH2PO4, 1.7 MgCl2, 20 HEPES, 11 glucose, 10 creatine, 20 taurine (pH = 7.4), and 21 mU/ml insulin and gassed with 100% O2. After 5 min of wash to eliminate the remaining blood, the heart was then perfused with fresh buffer mixed with 1.5 mg/ml collagenase type B (Boehringer Mannheim) for 8–15 min. The cells were then dispersed in a KB solution containing (in mmol/l) 70 K glutamate, 30 KCl, 10 KH2PO4, 1 MgCl2, 20 taurine, 10 glucose, and 10 HEPES.

Indirect immunofluorescent staining. Indirect immunostaining was performed as previously described (28, 29). Briefly, indirect immunofluorescent staining was performed on isolated rat cardiac myocytes with 4% paraformaldehyde for 20 min and was permeablized with 0.1% Triton X. The cells were quenched for aldehyde groups in 0.75% glycine buffer, washed extensively with PBS-Tween, and blocked with 5% normal goat sera for 2 h. The cells were incubated overnight at 4°C with primary anti-{alpha}1D antibody (1:200, Sigma). After extensive wash, the cells were then incubated with FITC-conjugated anti-rabbit antibody (1:200, Johnson Immunol). The cells were then washed with PBS-Tween and mounted in antifade mountant before being viewed with a confocal scanning laser microscope (MRC-600; Bio-Rad). Secondary antibody alone was used as control. {alpha}1D-, {beta}2a-, {alpha}2{delta}-cotransfected tsA201 and nontransfected cells were grown in cover slips and were also included in this study.

Expression of {alpha}1D Ca channel in tsA201 cells. tsA201 cells were grown and transiently transfected with 10 µg of a mixture of rat {alpha}1D, {beta}2a, and rabbit {alpha}2{delta} cDNAs (kindly provided by Dr. S. Seino from Keba University and Dr. E. Perez-Reyes from University of Virginia) in a molar ratio of 1:1:1, using the calcium phosphate method as previously described (1). After transfection, cells were maintained at 37°C. To identify transfected cells for patch-clamp studies, cells were cotransfected with an expression plasmid for a lymphocyte surface antigen (CD8-a). Two to three days after transfection, cells were incubated for 2 min in the intracellular solution containing anti-CD8 coated beads (Dynabeads M-450 CD8-a) (Dynal AS) (13). Transfected cells expressing CD8 on their surface are decorated with many beads and are thus readily distinguishable from nontransfected cells (13).

Recording of {alpha}1D ICa-L in tsA201 cells. Whole cell voltage-clamp recording was performed with the Axopatch 200B (Axon Instruments) with recording patch pipettes resistance of 1.5–2 M{Omega}. The internal solution contained (in mM) 135 CsCl, 4 MgCl2, 4 ATP, 10 HEPES, 10 EGTA, and 1 EDTA; adjusted to pH 7.2 with tetraethylammonium (TEA)-OH. The bath solution contains (in mM) 135 choline Cl, 1 MgCl2, 2 CaCl2, and 10 HEPES; adjusted to pH 7.4 with TEA-OH. Signals were sampled at 20 kHz and low pass filtered at 2 kHz. Data were leak subtracted on-line using the P/4 protocol and analyzed using pCLAMP V8.0 (Axon Instruments). Junction potentials were always compensated and were <5 mV. For {alpha}1D ICa-L current-voltage relations, tsA201 cells were depolarized from a holding potential of –100 mV to test potentials between –80 and 60 mV with increments of 10 mV. For the time course, {alpha}1D ICa-L was continuously recorded at a test potential of –10 mV from a holding potential of –100 mV.

Western blot. tsA201 cells were harvested by mechanical scrapping 48 h after transfection with {alpha}1D, {beta}2a, and {alpha}2{delta} cDNAs at basal and at stimulated conditions (0.3 mM 8-bromo-cAMP for 30 min before being harvested) (16). Cells were lysed in a lysis buffer [in mM: 50 Tris·HCl (pH = 7.4) 150 NaCl, 5 EDTA, 0.25% Triton-X 100, 10% glycerol, 1 NaF, and 1 Na3VO4, plus 10 mg/ml phenylmethylsulfonyl fluoride, aprotinin, and leupeptin]. Cell lysate was centrifuged at 14,000 g for 30 min. The supernatants were resolved by 4–12% SDS-PAGE (150 µg protein each lane). Phosphorylated {alpha}1D channels were detected by using the mouse antiphosphoserine antibodies (0.1 µg/ml, Biomol) and visualized by chemiluminescence with the ECL-plus Western blotting detection system (Amersham Pharmacia). An anti-{alpha}1D antibody (Sigma) was used to detect total {alpha}1D Ca channel protein in the lysate. Western blot images were scanned and analyzed using Sigma Gel. Intensity of the image using the antiphosphoserine antibody (recognizes only phosphorylated {alpha}1D Ca channel protein) was normalized to those obtained with the anti-{alpha}1D antibody (recognizes total {alpha}1D Ca channel protein) for each experiment to minimize possible effects of loading errors. The normalized signal for {alpha}1D phosphorylation was compared in the absence and in the presence of 8-bromo-cAMP. Similar experiments were also performed using an equal amount of membrane protein from 6-mo-old rat left and right atria (n = 4).

Statistic analysis. Statistical comparisons were evaluated using either paired or unpaired Student's t-test, as appropriate. Data are presented as means ± SE. A value of P < 0.05 is considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
{alpha}1D Ca channel transcript is present in rabbit SA node, AV node and in atria, but not in ventricles. Regional distribution of {alpha}1D Ca channel in adult rabbit hearts was investigated using RT-PCR. RT-PCR results (n = 3) showed that a band of 1.047 kb corresponding to {alpha}1D was amplified from rabbit SA node, atria, AV node, but not from the ventricle (Fig. 1). A 361-bp band corresponding to the S15 housekeeping gene was seen in all the tissues (Fig. 1, bottom), confirming the accuracy in RNA estimation and gel-loading techniques. In contrast to the ubiquitous expression of {alpha}1C Ca channel, the restricted expression pattern of {alpha}1D Ca channel in the heart supports its unique and potential roles in the SA node (19, 27, 43) and atrial tissue, respectively.



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  		Fig. 1. Expression of {alpha}1D Ca channel mRNA in adult rabbit heart by RT-PCR. With the use of a {alpha}1D Ca channel-specific primer, a transcript of 1047 bp corresponding to {alpha}1D Ca channel was amplified from the rabbit sinoatrial (SA) node (SAN), right atrium (RA), and atrioventricular (AV) node (AVN) but not from the right ventricle (RV) (top). A 361-bp transcript corresponding to housekeeping gene S15 was seen in all the tissues (bottom).

 
{alpha}1D Ca channel protein is present in rat atrial cells. The limitation of RT-PCR using total RNA isolated from cardiac tissue is the possible contamination with noncardiomyocytes, such as neuronal and/or vascular tissue. To exclude this possibility, indirect immunostaining using anti-{alpha}1D antibody was performed in isolated adult rat atrial and ventricular myocytes, as shown in Fig. 2. The reason for choosing rat myocytes over rabbit myocytes is that all the commercially available antibodies are raised in rabbit against the rat {alpha}1D Ca channel sequence. Anti-{alpha}1D antibody shows clear membrane staining in atrial (Fig. 2B) but not in ventricular myocytes (Fig. 2F). Similar observations were seen in another two independent experiments. This type of staining was not seen with the secondary antibody alone (Fig. 2D). The specificity of the anti-{alpha}1D Ca channel antibody was further tested on the {alpha}1D Ca channel in transfected and nontransfected tsA201 cells. Marked membrane staining was readily observed in the cells that were successfully transfected with {alpha}1D Ca channel (Fig. 3, A,a and B,b). No staining was observed in the neighboring tsA201 cells, which failed to express the channel (Fig. 3 A,a and B,b). In addition, there is no detectable staining on the nontransfected tsA201 cells under the same confocol scanning settings (Fig. 3D), providing the most compelling evidences for the specificity of the staining of the anti-{alpha}1D antibody on rat cardiac myoctyes (Fig. 2). We also investigated the level of expression of {alpha}1D Ca channel protein in the right versus left atria. The Western blot data shown in Fig. 4 demonstrate no significant difference in the amount of {alpha}1D Ca channel proteins in the right versus left rat atrium (Fig. 4A). Averaged data from a total of four experiments are shown in Fig. 4B.



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  		Fig. 2. Expression of {alpha}1D Ca channel protein in adult rat atrial but not in ventricular myocytes. Confocal indirect immunostaining experiments using anti-{alpha}1D Ca channel antibody were performed on freshly isolated adult rat cardiac myocytes. A, C, and E: phase controls. A: two overlapping atrial cells from an adult rat heart. B: corresponding staining with anti-{alpha}1D Ca channel antibody. Clear sarcolemmal staining was observed in these adult rat atrial myocytes. C: an atrial myocyte. D: no specific staining was observed using the secondary antibody alone, indicating the specificity of the staining pattern of anti-{alpha}1D antibody. E: two ventricular cells. F: corresponding staining with anti-{alpha}1D Ca channel antibody demonstrating the absence of any specific staining.

 


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  		Fig. 3. Confocol immunostaining of {alpha}1D Ca channel in transfected and nontransfected tsA201cells. A and B: phase controls and overlap of staining of the {alpha}1D-{beta}2a-{alpha}2{delta}-transfected tsA201 cells with anti-{alpha}1D Ca channel antibody. a and b: corresponding staining of the cells with anti-{alpha}1D antibody from different experiments. Note specific staining of {alpha}1D-transfected cells versus absence of staining on neighboring cells, which did not express the channel. C,c: no staining was observed with secondary antibody alone. D,d no staining of anti-{alpha}1D antibody on nontransfected tsA201 cells under the same confocol scanning setting. Similar results were observed in another two independent experiments.

 


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  		Fig. 4. Expression level of {alpha}1D Ca channel in rat right (RA) versus left atrium (LA) by Western blot analysis. Membrane protein (150 µg) from 6-mo-old rat LA and RA was loaded in each lane. Anti-{alpha}1D antibody was diluted 200 times and incubated overnight at 4°C. With the use of image analysis of the intensity of immunoblots, no significant difference of {alpha}1D Ca channel protein level was observed in RA versus LA (A). Averaged data from 4 experiments are shown in B.

 
Modulation of {alpha}1D ICa-L by PKA. PKA modulation of {alpha}1D ICa-L was determined in the mammalian expression system tsA201 cell line. This is mainly because both {alpha}1C and {alpha}1D Ca channels are expressed in the native myocytes (19, 27, 37, 43), and {alpha}1D ICa-L cannot be distinguished pharmacologically from {alpha}1C ICa-L because of the lack of selective blockers or agonists. Increased cAMP concentration was achieved by the use of cell-permeable 8-bromo-cAMP (33). In contrast to {alpha}1C-subunit of the cardiac Ca channel, which alone is sufficient to function as a voltage-gated Ca channel when expressed in heterologous cells (41), {alpha}1D Ca channel subunit alone failed to yield any functional current when expressed in tsA201 cells. This is consistent with reports from Xu and Lipscombe (39) and Bell et al. (2). Cotransfection of tsA201 with the {beta}2a-, {alpha}2{delta}- and {alpha}1D-subunits yielded a functional {alpha}1D ICa-L. {alpha}1D ICa-L was recorded from a holding potential of –100 mV with 2 mM Ca as charge carrier 48 h after transfection (Fig. 5A). Expressed {alpha}1D ICa-L activated between –60 and –50 mV and peaked at –10 mV (Fig. 5B). Application of 10 nM nifedipine partially blocked the expressed {alpha}1D ICa-L, whereas 10 µM almost completely abolish the current (Fig. 5C). No ICa-L was observed in {beta}2a + {alpha}2{delta}-transfected tsA201 cells (without {alpha}1D-subunit) (Fig. 5D), indicating that there is no functional endogenous {alpha}1D channel in tsA201 cells.



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  		Fig. 5. Expression and characterization of {alpha}1D Ca channel in tsA201 cells. A and B: current trances and averaged current-voltage (I-V) relationships in {alpha}1D-{beta}2a-{alpha}2{delta}-transfected tsA201 cells using 2 mM Ca as the charge carrier. Superimposed current traces activated by depolarizing pulses between –80 to 60 mV from a holding potential of –100 mV are shown. C: expressed {alpha}1D L-type Ca current (ICa-L) was blocked by a high concentration (10 µM) of nifedipine, a Ca channel blocker. D: no ICa-L was recorded in tsA201 cell transfected with {beta}2a/{alpha}2{delta}-subunits only.

 
Superfusion of cells with 50 µM 8-bromo-cAMP significantly enhanced the channel activity (Fig. 6). Averaged data show that 8-bromo-cAMP increased {alpha}1D ICa-L from 35.1 ± 7.4 to 63.6 ± 13.4 pA/pF (78.5 ± 19%, P < 0.05, n = 7) without shift of current-voltage relationships (Fig. 6). No significant difference in the steady-state inactivation was observed before [half-maximal voltage (V1/2) = –33.5 ± 0.36 mV; k = 7.26 ± 0.32, n = 7] and upon cAMP-dependent stimulation (V1/2 = –34.6 ± 0.53 mV; k = 6.6 ± 0.55, n = 7) (Fig. 6). Specificity of PKA effect on {alpha}1D ICa-L enhancement was tested using a cell-permeable PKA inhibitor peptide (PKI), which is known to be highly specific for PKA over other kinases. Application of 50 µM PKI reversed the cAMP stimulatory effect (Fig. 7). However, the basal amplitude of {alpha}1D ICa-L was not affected after PKI application (Fig. 7).



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  		Fig. 6. 8-Bromo-cAMP (8-Br-cAMP) increases {alpha}1D ICa-L expressed in tsA 201 cells. {alpha}1D, together with {beta}2a/{alpha}2{delta}-subunits, were cotransfected in tsA201 cells. Whole cell {alpha}1D ICa-L was recorded using 2 mM Ca as charge carrier. A: time course of {alpha}1D ICa-L before and after application of 50 µM 8-Br-cAMP. Current amplitudes were normalized for easy comparison. Inset, selected peak current traces. B: averaged I-V curves before and after application of 8-Br-cAMP (n = 7). C: steady-state inactivation of {alpha}1D ICa-L before and after application of 8-Br-cAMP. D: averaged data of the increase of expressed {alpha}1D ICa-L in tsA 201 cells by 50 µM 8-Br-cAMP (n = 7, P < 0.05). *Significance of P < 0.05.

 


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  		Fig. 7. Increase of expressed {alpha}1D ICa-L by 8-Br-cAMP was blocked by specific protein kinase A (PKA) inhibitor (PKI). {alpha}1D ICa-L was recorded from a holding potential of –100 mV using 2 mM Ca as charge carrier at 48 h after transfection of tsA201 cells with {alpha}1D-{beta}2a-{alpha}2{delta} plasmids. Superfusion of 50 µM of cell-permeable 8-Br-cAMP resulted in a 75% increase in {alpha}1D ICa-L in one cell. This increase was prevented by the addition of the membrane-permeable PKI (50 µM) to 8-Br-cAMP.

 
Phosphorylation of {alpha}1D channel protein by PKA. The functional data above showed that activation of PKA by 8-bromo-cAMP significantly increased {alpha}1D ICa-L. To determine whether the phosphorylation of {alpha}1D channel in the cellular context is responsible for the increased channel activity by PKA, Western blot was performed using proteins from transfected tsA201 cells under basal condition and cells treated with 8-bromo-cAMP. Phosphorylation of {alpha}1D Ca channel was detected by immunoblot with the antiphosphoserine antibody. Total {alpha}1D Ca channel protein was detected using anti-{alpha}1D antibody. Anti-{alpha}1D antibody recognized the 190-kDa band, corresponding to the {alpha}1D Ca channel protein. Antiphosphoserine antibodies detected the 190-kDa band of phosphorylated {alpha}1D Ca channel proteins in the 8-bromo-cAMP-treated tsA201 cells (Fig. 8). With the use of image analysis of the intensity of immunoblots for phosphorylated {alpha}1D corrected for loading error by total {alpha}1D Ca channel intensity, exposure of transfected tsA201 cells to 8-bromo-cAMP increased {alpha}1D Ca channel phosphorylation by fivefold (n = 5, P < 0.05). These results demonstrate that PKA phosphorylation of the {alpha}1D Ca channel is consistent with increased {alpha}1D ICa-L observed in the presence of 8-bromo-cAMP.



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  		Fig. 8. Phosphorylation of {alpha}1D Ca channel by PKA. Western blot was performed using proteins extracted from {alpha}1D-{beta}2a-{alpha}2{delta}-transfected tsA201 cells under basal and stimulated (0.3 mM 8-Br-cAMP, 30 min) conditions. A: phosphorylation of {alpha}1D channel was detected with the antiphosphoserine antibodies. Total {alpha}1D protein was detected using anti-{alpha}1D antibody. Antiphosphoserine antibodies detected the 190-kDa band of phosphorylated {alpha}1D Ca channel protein from the 8-Br-cAMP-treated tsA201 cells. B: with the use of image analysis of the intensity of immunoblots for phosphorylated {alpha}1D Ca channel protein corrected for loading error by total {alpha}1D Ca channel protein intensity, exposure to 8-Br-cAMP increased {alpha}1D phosphorylation by fivefold (n = 5, P < 0.05). *Significance P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present data establish that {alpha}1D L-type Ca channel expression is restricted to the rabbit SA node, AV node, and atria but is not expressed in adult ventricles. This observation is in agreement with reports on rat by Takimoto et al. (37) and on mice and human heart by Mangoni et al. (19), indicating that the SA expression of the {alpha}1D Ca channel is not species dependent. The restricted expression of {alpha}1D Ca channel in the heart suggests its unique role in SA tissue. Significant intrinsic sinus bradycardia in both isolated heart and in the whole animal has been reported in {alpha}1D Ca channel knockout mice (19, 27, 43). Furthermore, SA node cells from {alpha}1D Ca channel knockout mice showed a significant increase of action potential cycle length compared with wild-type mice (19, 43). Altogether, these data establish the significant role of the {alpha}1D Ca channel in the pacemaker activity of the SA node. The fact that the {alpha}1D Ca channel transcript was expressed in atria, but not in ventricles, also evokes the important potential role of the Ca channel in atria under both physiological and pathological settings such as atrial fibrillation (AF). L-type Ca channel can play a key role in the initiation and persistence of AF (3). Whole cell voltage-clamp studies in canine atrial cardiomyocytes showed that atrial tachycardia leads to a substantial reduction in ICa-L density (24), and 60% reduction of ICa-L in AF was reported in human atrial cardiomyocytes (38). There is also growing focus on the importance of electrophysiological heterogeneity between both atria in the initiation and maintenance of AF (11, 18, 21, 32). In this study, we show that there are no significant differences in the protein level of the {alpha}1D Ca channel from the left and right atria, suggesting minor contribution of {alpha}1D ICa-L in the interatrial ionic current heterogeneity. Nevertheless, the exclusive expression of {alpha}1D Ca channel in atria, but not in ventricles, makes {alpha}1D Ca channel an attractive target for developing novel therapies for atrial diseases such as AF.

The phosphorylation process constitutes one of the major regulatory pathways for cardiac Ca channels. Sympathetic stimulation will activate {beta}-adrenergic pathway, which in turn stimulates adenylate cyclase via a G protein resulting in the production of cAMP (22). As mentioned before, SA tissue is heavily innervated with the SNS (30), and cAMP-PKA is also the converging point of regulation of Ca channels by many neurotransmitters and hormone receptors, including the serotoninergic 5-HT4 receptor and {beta}1- and {beta}2-adrenoreceptors, which are identified in human atrial cells (8, 14, 20, 25, 35, 40). The results in this study, together with previous reports (2, 19, 37, 39) established the coexistence of {alpha}1C and {alpha}1D Ca channels in both the SA node and atria. It has been well established that PKA activation greatly enhances the {alpha}1C Ca channel activity (22). It is therefore fundamental to investigate whether cAMP-PKA also modulates the {alpha}1D Ca channel. The present data showed that {alpha}1D Ca channel is under the specific modulation by PKA. We showed for the first time that activation of PKA by 8-bromo-cAMP significantly increased the expressed {alpha}1D ICa-L by phosphorylation of the {alpha}1D Ca channel subunit in tsA201 cells. The {beta}2a-subunit has also been reported to be the substrate of PKA phosphoryaltion (5, 16). The role of {beta}2a in the modulation of {alpha}1D ICa-L expressed in tsA201 cells by 8-bromo-cAMP cannot be excluded. The {alpha}1D-subunit has been shown to possess multiple potential PKA phosphorylation sites (23); however, the exact phosphorylation site(s) is not known and warrants further investigations.

As mentioned above, cAMP-dependent regulation of the cardiac L-type Ca channel is well established in native cells. However, this is not the case in the heterologous expression systems. Both an increase (40, 41) and no effect (26, 34, 44) of the expressed {alpha}1C ICa-L have been reported upon PKA activation. This discrepancy in channel regulation between native cells and heterologous expression systems suggest that critical factors involved in the regulation of Ca channel by PKA are absent in the expression systems. For example, A kinase anchoring proteins have been shown to facilitate the phosphorylation and regulation of the {alpha}1C Ca channel on PKA activation. Coexpression of A kinase anchoring proteins with the {alpha}1C Ca channel restores the PKA-dependent increase of {alpha}1C ICa-L in heterologous expression systems (9, 12). Under our experimental conditions using tsA201 cells, {alpha}1D ICa-L was successfully increased by 8-bromo-cAMP, indicating different regulatory mechanism from the {alpha}1C Ca channel.

In conclusion, characterization of the expression and modulation of the {alpha}1D L-type Ca channel in the heart will enhance our knowledge of its physiological function and potential role in pathological settings, thereby leading to the development of new therapeutic approaches to manage supraventricular arrhythmias.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work is supported by a Veteran Affairs Merit grant, National Heart, Lung, and Blood Institute Grant R01 HL-077494, and Research Enhancement Award Program (REAP) to M. Boutjdir. Veterans Integrated Service Network (VISN3) Seed Grant and Veteran Affairs Merit Review Entry Program (MREP) grant to Y. Qu. The Canadian Institutes of Health Research award to Dr. G. Baroudi.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Boutjdir, Research and Development (151A), VA New York Harbor Healthcare System, 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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