Unchanged β-Adrenergic Stimulation of Cardiac L-type Calcium Channels in Cav1.2 Phosphorylation Site S1928A Mutant Mice
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m804981200
ISSN1083-351X
AutoresToni Lemke, Andrea Welling, Carl J. Christel, Anne Blaich, Dominik Bernhard, Peter Lenhardt, Franz Hofmann, Sven Moosmang,
Tópico(s)Receptor Mechanisms and Signaling
ResumoPhosphorylation of serine 1928 (Ser1928) of the cardiac Cav1.2 subunit of L-type Ca2+ channels has been proposed as the mechanism for regulation of L-type Ca2+ channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser1928 to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the β-adrenergic receptor, whereas the mutation abolished phosphorylation of Cav1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser1928 of Cav1.2 is not functionally involved in β-adrenergic stimulation of Cav1.2-mediated Ca2+ influx into the cardiomyocyte. Phosphorylation of serine 1928 (Ser1928) of the cardiac Cav1.2 subunit of L-type Ca2+ channels has been proposed as the mechanism for regulation of L-type Ca2+ channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser1928 to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the β-adrenergic receptor, whereas the mutation abolished phosphorylation of Cav1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser1928 of Cav1.2 is not functionally involved in β-adrenergic stimulation of Cav1.2-mediated Ca2+ influx into the cardiomyocyte. There is excellent evidence for a regulation of the cardiac L-type Ca2+ current (ICaL) by β-adrenoceptors, cAMP, and protein kinase A (PKA) 2The abbreviations used are: PKA, protein kinase A; ECG, electrocardiogram; FS, fractional shortening; tk, thymidine kinase; PKAc, catalytic PKA; Ctr, control; Iso, isoproterenol. (1.Osterrieder W. Brum G. Hescheler J. Trautwein W. Flockerzi V. Hofmann F. Nature. 1982; 298: 576-578Crossref PubMed Scopus (256) Google Scholar, 2.Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1958) Google Scholar). This mechanism most likely plays a critical role in physiological processes in the heart, e.g. excitation-contraction coupling, the regulation of inotropy and chronotropy, as well as pathological processes such as heart failure (for review see Refs. 3.Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3397) Google Scholar, 4.Bodi I. Mikala G. Koch S.E. Akhter S.A. Schwartz A. J. Clin. Investig. 2005; 115: 3306-3317Crossref PubMed Scopus (232) Google Scholar, 5.Kamp T.J. Hell J.W. Circ. Res. 2000; 87: 1095-1102Crossref PubMed Scopus (502) Google Scholar). The molecular basis of ICaL regulation by PKA could not be defined conclusively so far (5.Kamp T.J. Hell J.W. Circ. Res. 2000; 87: 1095-1102Crossref PubMed Scopus (502) Google Scholar). This is mainly due to the fact that the extent of regulation of ICaL activity by PKA in experiments in transfected cells (10–50% increase) falls well short of the magnitude recorded in native cardiac cells (200–400% increase). It seems likely that regulatory influences not reproduced in heterologous expression systems are important for the control of the activity of cardiac Ca2+ channels in vivo (2.Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1958) Google Scholar). It is a widely accepted finding that phosphorylation of Ser1928 of the Cav1.2 L-type channel subunit is necessary for β-adrenergic regulation of ICaL (5.Kamp T.J. Hell J.W. Circ. Res. 2000; 87: 1095-1102Crossref PubMed Scopus (502) Google Scholar, 6.Hulme J.T. Westenbroek R.E. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16574-16579Crossref PubMed Scopus (129) Google Scholar). The amino acid Ser1928, which is located in the intracellular C terminus of Cav1.2, has been reported to be the only detectable in vivo and in vitro PKA phosphorylation site of the Cav1.2 subunit (6.Hulme J.T. Westenbroek R.E. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16574-16579Crossref PubMed Scopus (129) Google Scholar, 7.Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. Science. 2001; 293: 98-101Crossref PubMed Scopus (444) Google Scholar, 8.Davare M.A. Hell J.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 16018-16023Crossref PubMed Scopus (111) Google Scholar, 9.De Jongh K.S. Murphy B.J. Colvin A.A. Hell J.W. Takahashi M. Catterall W.A. Biochemistry. 1996; 35: 10392-10402Crossref PubMed Scopus (244) Google Scholar, 10.Hall D.D. Feekes J.A. Arachchige Don A.S. Shi M. Hamid J. Chen L. Strack S. Zamponi G.W. Horne M.C. Hell J.W. Biochemistry. 2006; 45: 3448-3459Crossref PubMed Scopus (100) Google Scholar). Electrophysiological studies using heterologous expression of the Cav1.2 subunit reported contrary findings, i.e. stimulation (11.Sculptoreanu A. Rotman E. Takahashi M. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10135-10139Crossref PubMed Scopus (164) Google Scholar) or no stimulation (12.Zong X. Schreieck J. Mehrke G. Welling A. Schuster A. Bosse E. Flockerzi V. Hofmann F. Pfluegers Arch. 1995; 430: 340-347Crossref PubMed Scopus (80) Google Scholar) of ICaL by PKA when only the Cav1.2 subunit was expressed. In contrast, the necessity of coexpression and phosphorylation of the Cavβ2a subunit was reported (13.Bunemann M. Gerhardstein B.L. Gao T. Hosey M.M. J. Biol. Chem. 1999; 274: 33851-33854Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Recently, the functional importance of Cavβ2a phosphorylation for β-adrenergic regulation of ICaL has been questioned (14.Miriyala J. Nguyen T. Yue D.T. Colecraft H.M. Circ. Res. 2008; 102: E54-E64Crossref PubMed Scopus (53) Google Scholar). Regardless of these findings, mutation of Cav1.2 Ser1928 to alanine prevented phosphorylation and regulation of the channel by PKA in heterologous expression systems (15.Gui P. Wu X. Ling S. Stotz S.C. Winkfein R.J. Wilson E. Davis G.E. Braun A.P. Zamponi G.W. Davis M.J. J. Biol. Chem. 2006; 281: 14015-14025Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 16.Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 17.Oliveria S.F. Dell'Acqua M.L. Sather W.A. Neuron. 2007; 55: 261-275Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). In contrast to these reports, Ganesan et al. (18.Ganesan A.N. Maack C. Johns D.C. Sidor A. O'Rourke B. Circ. Res. 2006; 98: E11-E18Crossref PubMed Scopus (89) Google Scholar) postulated that at least 70% of the β-adrenergic regulation of ICaL in virally transducted heart cells cannot be attributed to the Ser1928 phosphorylation event. However, the viral infection system by Ganesan et al. (18.Ganesan A.N. Maack C. Johns D.C. Sidor A. O'Rourke B. Circ. Res. 2006; 98: E11-E18Crossref PubMed Scopus (89) Google Scholar) only partially reconstituted the regulation of ICaL (the PKA-mediated increase in current was only 50%). On the other hand, Hulme et al. (6.Hulme J.T. Westenbroek R.E. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16574-16579Crossref PubMed Scopus (129) Google Scholar) recently correlated β-adrenergic stimulation with phosphorylation of Ser1928 and functional up-regulation of ICaL in intact ventricular myocytes. In addition to these findings, Oliveria et al. (17.Oliveria S.F. Dell'Acqua M.L. Sather W.A. Neuron. 2007; 55: 261-275Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) postulated a critical role for the Ser1928 phosphorylation in the PKA-mediated regulation of Cav1.2 channels in HEK293 cells and neurons. To analyze this controversial issue in intact, untransfected, native cardiomyocytes containing the complete regulatory system, we generated a knock-in mouse line carrying the Cav1.2S1928A mutation. Generation of Mice Lacking the Ser1928 Phosphorylation Site on Cav1.2—To construct the targeting vector, a 7.4-kb fragment containing exons 44–47 of CACNA1C was isolated from 129/Sv mouse genomic DNA. The targeting vector included a 1.2-kb short arm and 6.2-kb long arm with PGK-neo and the thymidine kinase gene (tk) flanked by two loxP sites. The 3′-side long arm contained exon 45 with the phosphorylation site, serine 1928, mutated to alanine. All mutation procedures were carried out by overlap PCR mutagenesis. The targeting construct was electroporated into R1 ES cells (129/Sv×129/Sv-CP F1) (19.Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1992) Google Scholar). Positive clones were identified by PCR and confirmed by Southern blot using an outer probe (5′-probe in Fig. 1a). Two positive clones were transfected with a Cre-expressing plasmid to delete the neo/tk marker genes. Five clones with the deletion event were injected in C57BL/6 blastocysts, and chimeras were crossed to C57BL/6 mice. Heterozygous mice were bred to produce homozygotes. The intercross of heterozygotes resulted in production of wild type, heterozygous, and homozygous offspring at almost the expected Mendelian ratio (125:215:88). For all analyses, mice with 129/Sv and C57BL/6 hybrid genetic background (Cav1.2S1928A-129B6F2) were used. All procedures relating to animal care and treatment conformed to the institutional and Regierung von Oberbayern guidelines. Preparation and Solubilization of Membranes and GST Fusion Proteins—All preparative steps were performed at 4 °C using precooled solutions containing the protease inhibitor mixture (2 μl/ml; Sigma), phenylmethylsulfonyl fluoride (200 mmol/liter), calpain inhibitor I (8 μg/ml), and calpain inhibitor II (8 μg/ml). Hearts from adult mice were frozen and pulverized under liquid N2 in a porcelain mortar and then resuspended in membrane preparation buffer containing 300 mmol/liter sucrose, 75 mmol/liter NaCl, 20 mmol/liter EDTA, 20 mmol/liter EGTA, 10 mmol/liter Tris-HCl, pH 7.4 (1 ml/100 mg of tissue). Homogenates were centrifuged two times at 4,500 rpm for 5 min at 4 °C to remove larger cell fragments, including nuclei. Membranes were collected by ultracentrifugation (50,000 rpm at 4 °C) for 30 min, and channels were solubilized for 20 min on ice with 1% deoxycholate, 10 mmol/liter EDTA, 10 mmol/liter EGTA, 50 mmol/liter Tris-HCl, pH 7.4, containing protease inhibitors. Nonsoluble material was removed by a second ultracentrifugation step (50,000 rpm at 4 °C for 30 min). GST fusion proteins were expressed in BL21 Escherichia coli according to the manufacturer's instructions (Amersham Biosciences). Antibodies—The anti-Cav1.2 and -Cavβ2a antibodies have been described previously (20.Moosmang S. Schulla V. Welling A. Feil R. Feil S. Wegener J.W. Hofmann F. Klugbauer N. EMBO J. 2003; 22: 6027-6034Crossref PubMed Scopus (248) Google Scholar, 21.Ludwig A. Flockerzi V. Hofmann F. J. Neurosci. 1997; 17: 1339-1349Crossref PubMed Google Scholar). Antibodies against the catalytic (PKAc) and regulatory subunits (RIIα and RIIβ) of cAMP-dependent protein kinase were purchased from BD Biosciences. The antibodies against GST and against the β1-adrenergic receptor were obtained from Calbiochem and Upstate, respectively. The phosphospecific antibody against Ser(P)1928 was generated by CovalAb against the peptide NH2-CLGRRA(pS)FHLECLK-COOH. The sensitivity and specificity of the phospho-specific antibody were confirmed utilizing GST fusion proteins/enzyme-linked immunosorbent assay/incubation with phosphorylated antigenic peptide (CovalAb). Immunoprecipitation and Immunoblotting—Thesolubilizedmembranes from heart were preincubated with protein A-Sepharose (Sigma) to remove proteins that bind to the resin nonspecifically. After removal of the Sepharose beads by centrifugation, the supernatant was incubated on ice with antibodies. After 2.5 h, protein A-Sepharose was added; samples were tilted for 1 h, and the resins were washed and extracted with 1:6 (v/v) SDS sample buffer (22.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). For total cellular protein analysis, hearts were homogenized in lysis buffer (2% SDS, 50 mmol/liter, Tris, pH 7.4). Proteins were separated on 10% SDS-polyacrylamide gels, blotted, and probed with antibodies by using a chemiluminescence detection system or nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as described in Ref. 20.Moosmang S. Schulla V. Welling A. Feil R. Feil S. Wegener J.W. Hofmann F. Klugbauer N. EMBO J. 2003; 22: 6027-6034Crossref PubMed Scopus (248) Google Scholar. To quantitatively evaluate Ser1928 phosphorylation, blots were first probed with anti-phospho-Ser1928 antibody and subsequently with anti-Cav1.2 antibody to correct for variability in the amount of total Cav1.2. The ratio of the anti-phospho-Ser1928 antibody to the anti-Cav1.2 signal was determined for each sample by blot densitometry using Quantity One software (Bio-Rad). Ratios were normalized to the ratio of the control animals. Phosphorylation with PKA—For PKA phosphorylation reactions, full-length Cav1.2 was immunoprecipitated with the Cav1.2-specific antibody, and immune complexes were captured on protein A-Sepharose. GST fusion proteins were purified on glutathione-Sepharose. Precipitated complexes were resuspended in phosphorylation buffer (0.1% Triton X-100, 50 mmol/liter HEPES-NaOH, pH 7.4, 10 mmol/liter MgCl2, 0.5 mmol/liter EGTA, 0.5 mmol/liter dithiothreitol). Phosphorylation was carried out by mixing the Sepharose pellets with 33 μmol/liter Mg-[γ-32P]ATP (6.6 μCi/reaction). The reaction, initiated by the addition of 5 units of PKAc (Sigma) to either the GST fusion proteins or immunoprecipitated Cav1.2 complexes, was carried out at 23 °C for 5 min (GST fusion proteins) or 10 min (immunoprecipitated Cav1.2 complexes) and terminated by boiling in 1:6 (v/v) SDS sample buffer. Importantly, phosphorylation without the addition of exogenous PKAc yielded no detectable autoradiography signals under these conditions. Proteins were separated on 10 or 12.5% SDS gels, and incorporated 32Pi was detected by autoradiography. To quantitatively evaluate the autoradiography signals, the ratio of the intensity of each signal was determined for each sample using a Fujix Bas1000 PhosphorImager and Aida 2.11 software. Signal intensities were normalized to the signal in the control preparations. Cell Isolation—Ventricular myocytes were isolated as described (AfCS Procedure Protocol PP00000125), maintained at 37 °C, and aerated with 98% O2, 2% CO2. Electrophysiology—Calcium currents (ICa) were recorded in whole-cell mode at room temperature from rod-shaped, striated, calcium-tolerant myocytes within 1–24 h of isolation. The extracellular solution contained 140 mm tetraethylammonium·Cl, 2 mm MgCl2, 1.8 mm CaCl2, 10 mm HEPES, and 10 mm glucose, pH 7.4. Patch pipettes (1–2 megohms) were filled with an intracellular solution, pH 7.4, containing 135 mm CsCl, 1 mm MgCl2, 5 mm MgATP, 10 mm HEPES, and 10 mm 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Recordings were discarded if the series resistance was over 6 megohms. Calcium current was elicited by either repeated 200-ms depolarizing pulses to 10 mV or a series of depolarizing pulses to different test potentials (–40 to +80 mV in 10-mV steps) from a holding potential of –40 mV. Calcium current was measured as the difference between the peak inward current and the current at the end of the test pulse. After establishing a solid baseline, the effects of either isoproterenol (100 nmol/liter) containing an equal concentration of ascorbic acid or forskolin (10 μmol/liter) on ICa were examined. H89 (Sigma), ICI118551 (Tocris), and CGP20712A (Tocris) were diluted from stocks in DMSO (H89, 10 mm) and water (10 mm, respectively). Currents were recorded with a patch clamp EPC9 device (HEKA, Lambrecht, Germany) and sampled at 5 kHz. Data acquisition and command potentials were controlled by Pulse+Pulsefit software version 8.54 (HEKA, Lambrecht, Germany), and data were stored for later off-line analysis. Leak compensation was performed online in Pulse+Pulsefit when necessary. All data are expressed as the means ± S.E. Values of p < 0.05 were considered significant. Telemetric Electrocardiogram (ECG) Recordings—Radiotelemetric ECG transmitters (ETA-F20, DSI, St. Paul, MN) were implanted into the peritoneal cavity under general anesthesia with isoflurane/O2. The ECG leads were sutured subcutaneously onto the upper right chest muscle and the upper left abdominal wall muscle. The animals were allowed to recover for 2 weeks before the experiments. Isoproterenol (Sigma) was dissolved in 0.9% NaCl. After 15 min of base-line recording, the mice were injected intraperitoneally with the drugs used. The ECGs were recorded for 45 min thereafter. The animals were allowed to recover for at least 48 h between experiments. Data were acquired using the DSI acquisition system. Open Field—The open field consisted of a transparent plastic box with a white floor (41 × 41 × 41 cm). The illumination at floor level was 150 lux. Mice were individually placed into the center of the open field, and their behaviors were tracked with an automated activity monitoring system (TSE Systems GmbH, Germany). The overall distance traveled by the mice and the vertical plane entries (rearings) was monitored for 5 min. Beam Walking—The beam consisted of long strips of plastic (1 m) with a 1.0-cm cross-section and grooves (0.5 × 1.0 cm) every 5 cm. The beam was placed horizontally, 50 cm above the bench surface, with both ends mounted on a narrow support. During training mice were placed at the start of the beam and trained once to traverse the beam. 24 h later the number of times the hind feet slipped off the beam was recorded. To detect motor learning, mice had to traverse the beam after 1 h and again after 24 h. Echocardiography—Images were obtained using a Vevo 770 Visual Sonics scanner equipped with a 30-MHz probe (Visual Sonics Inc., Toronto, Canada). The mice were lightly anesthetized (1.5% isoflurane), anchored to a warming platform in dorsal position, and ECG limb electrodes were placed. The chests were shaved and cleaned to minimize ultrasound attenuation. Fractional shortening (FS, reduction of the length of the end-diastolic diameter that occurs by the end of systole) was assessed from the M mode of the parasternal short axis view. Ctr and Ki mice were studied before and 1 min after administration of isoproterenol (0.5 mg/kg body weight intraperitoneally). We used a gene-targeting strategy that utilized a replacement vector containing the point mutation and a neo/tk gene cassette flanked by loxP sites (Fig. 1a). All homozygous mutants analyzed were F2 mice from a cross between the chimeras (contributing 129 background) and C57BL/6 mice (Cav1.2S1928A-129B6F2). The mutants showed no overt cardiovascular phenotype and bred normally. Cav1.2S1928A-129B6F2 animals could not be differentiated from litter-matched wild type mice in open field, and beam walking tests (data not shown), indicating no obvious changes in behavior or motor performance. We confirmed the mutation of the phosphorylation site by DNA sequencing, Southern blot (Fig. 1b), and immunoblot analysis. The point mutation and the loxP site did not alter the expression of the Cav1.2 protein (Fig. 1c). The Cav1.2S1928A-129B6F2 mutation did not decrease the expression of β1-adrenoceptors or the catalytic and regulatory PKA subunits PKAc, RIIα, and RIIβ in the mutants. Coimmunoprecipitation demonstrated that the macromolecular complex between Cav1.2, PKAc, RIIα, and RIIβ (7.Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. Science. 2001; 293: 98-101Crossref PubMed Scopus (444) Google Scholar, 23.Balijepalli R.C. Foell J.D. Hall D.D. Hell J.W. Kamp T.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7500-7505Crossref PubMed Scopus (328) Google Scholar) was preserved (Fig. 1d). Using GST fusion proteins, we corroborated that mutation of Ser1928 to alanine in the C terminus of the Cav1.2 results in a complete loss of cAMP-mediated phosphorylation in vitro (Fig. 2a). Next, we developed a phospho-specific antibody to detect Ser1928 phosphorylation. In a PKA phosphorylation assay of GST fusion proteins, anti-phospho-Ser1928 phosphorylated at Ser1928. To extend these in vitro data to the Cav1.2S1928A-129B6F2 animal model, we next evaluated phosphorylation of cardiac Cav1.2 subunits from these mice. Phosphorylation by PKA in the presence of [γ-32P]ATP and the catalytic subunit of PKA (5 units) was examined using an immunoprecipitation protocol and autoradiography. Only weak phosphoprotein signals (9.4 ± 4.8% of control level) corresponding to Cav1.2 were observed when heart preparations from Cav1.2S1928A-129B6F2 mutants were used, whereas in controls Cav1.2 phosphorylation could be readily detected (Fig. 2c). In strong agreement with these results, detection of Cav1.2 by the phosphospecific antibody to Ser1928 was barely detectable in the homozygotes (4.4 ± 2.1% of control level). Specificity of the anti-Ser1928 antibody in Cav1.2 full-length preparations was additionally confirmed by preincubation with phosphorylated antigenic peptide, which abolished the immunoblot signal (Fig. 2d). Taken together, these findings suggest that Ser1928 is the only easily detectable Cav1.2 PKA phosphorylation site. Moreover, the Cav1.2 subunit lacking this phosphorylation site is properly targeted to the Cav1.2-associated proteins (7.Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. Science. 2001; 293: 98-101Crossref PubMed Scopus (444) Google Scholar, 23.Balijepalli R.C. Foell J.D. Hall D.D. Hell J.W. Kamp T.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7500-7505Crossref PubMed Scopus (328) Google Scholar), indicating that the phosphorylation site may not be critical for maintaining the steady-state level of channels at the membrane or the assembly of the Cav1.2 signaling complex. Next, we looked at whether the lack of this phosphorylation site on Cav1.2 affects β-adrenergic regulation in adult littermate controls and homozygotes. The current-voltage relationship and expression level measured as current density of the mutant channel were similar to that of the wild type channel (Fig. 3, a and c) indicating that L-type channels show no different voltage dependence for activation or membrane expression in Cav1.2S1928A-129B6F2 mice. To quantitatively correlate phosphorylation of Ser1928 with regulation of ICaL, we measured β-adrenergic stimulation of whole-cell ICaL by isoproterenol (Iso). Iso treatment increased ICaL in control mice 280 ± 25% and in Cav1.2S1928A-129B6F2 mice 268 ± 20%, respectively (Fig. 3, a and b). In addition to the unchanged Iso-induced increase in peak inward ICaL, the 10-mV left shift in the V½ of activation was preserved in cardiomyocytes from Cav1.2S1928A-129B6F2 mice (Fig. 3a). In agreement with the Iso results, we found that the adenylate cyclase agonist forskolin enhanced ICaL in both control and mutant cardiomyocytes to identical levels (228 ± 39% versus 240 ± 20%, see Fig. 4, a and b). Application of forskolin completely prevented further isoproterenol stimulation in both genotypes (Fig. 4, a and b), indicating that a membrane-delimited pathway involving the “direct” stimulation of ICaL by a Gs subunit (24.Hartzell H.C. Mery P.F. Fischmeister R. Szabo G. Nature. 1991; 351: 573-576Crossref PubMed Scopus (188) Google Scholar, 25.Yatani A. Codina J. Imoto Y. Reeves J.P. Birnbaumer L. Brown A.M. Science. 1987; 238: 1288-1292Crossref PubMed Scopus (362) Google Scholar) was not operative. To further substantiate these findings, we characterized the functional role of additional components of the β-adrenergic pathway in ICaL regulation. The PKA inhibitor H89 consistently blocked the isoproterenol-induced increase in ICaL in litter-matched control and Cav1.2S1928A-129B6F2 cardiomyocytes (Fig. 5, a and b). The isoproterenol effect was also completely suppressed by application of 0.1 μm of the β1-adrenergic blocker CGP20712A and partially by 0.1 μm of the β2-adrenergic blocker ICI118551 in wild type and mutant mice (Fig. 5c). Taken together, these data rule out a switch of the physiological β-adrenergic regulation of cardiac Cav1.2 in Cav1.2S1928A-129B6F2 mice. The results shown so far did not rule out the possibility that phosphorylation of Ser1928 is required for the in vivo regulation of cardiac function. First, we looked at a potential role of Cav1.2 Ser1928 phosphorylation in the regulation of heart rate and rhythm using ECG telemetry. There were no detectable differences in basal heart rate (587 ± 24 beats/min versus 567 ± 27 beats/min, t test, p > 0.05). Isoproterenol infusion increased heart rate to the same level (734 ± 15 beats/min versus 741 ± 6 beats/min, t test, p > 0.05) in both control and Cav1.2S1928A-129B6F2 animals (Fig. 6a). We next tested the consequence of ablation of Cav1.2 Ser1928 phosphorylation for cardiac contractility. Echocardiography clearly showed that isoproterenol increased cardiac FS, an indicator of systolic performance, to the same level in both genotypes (Fig. 6, b and c) demonstrating that the positive inotropic effect of β-adrenergic stimulation in vivo is not dependent on Ser1928. Taken together, our results support the notion that PKA phosphorylation of Ser1928 of Cav1.2 is functionally not involved in β-adrenergic regulation of ICaL in murine ventricular cardiomyocytes. The results are in line with the previous finding that β-adrenergic stimulation requires a PKA-mediated phosphorylation step (1.Osterrieder W. Brum G. Hescheler J. Trautwein W. Flockerzi V. Hofmann F. Nature. 1982; 298: 576-578Crossref PubMed Scopus (256) Google Scholar, 6.Hulme J.T. Westenbroek R.E. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16574-16579Crossref PubMed Scopus (129) Google Scholar, 9.De Jongh K.S. Murphy B.J. Colvin A.A. Hell J.W. Takahashi M. Catterall W.A. Biochemistry. 1996; 35: 10392-10402Crossref PubMed Scopus (244) Google Scholar, 11.Sculptoreanu A. Rotman E. Takahashi M. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10135-10139Crossref PubMed Scopus (164) Google Scholar, 13.Bunemann M. Gerhardstein B.L. Gao T. Hosey M.M. J. Biol. Chem. 1999; 274: 33851-33854Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 16.Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 18.Ganesan A.N. Maack C. Johns D.C. Sidor A. O'Rourke B. Circ. Res. 2006; 98: E11-E18Crossref PubMed Scopus (89) Google Scholar, 24.Hartzell H.C. Mery P.F. Fischmeister R. Szabo G. Nature. 1991; 351: 573-576Crossref PubMed Scopus (188) Google Scholar). Many questions remain to understand the β-adrenergic regulation of Ca2+ channels. What is the physiological substrate of PKA in the Cav1.2 channel complex responsible for acute stimulation of ICaL, if not Ser1928? The phosphorylation target could be the Cavβ2a subunit (13.Bunemann M. Gerhardstein B.L. Gao T. Hosey M.M. J. Biol. Chem. 1999; 274: 33851-33854Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) or the giant protein AHNAK (26.Haase H. Alvarez J. Petzhold D. Doller A. Behlke J. Erdmann J. Hetzer R. Regitz-Zagrosek V. Vassort G. Morano I. FASEB J. 2005; 19: 1969-1977Crossref PubMed Scopus (65) Google Scholar). Our studies provide the basis to address these critical questions in the future. Because it has been difficult to reconstitute reproducibly ICaL regulation in cultured cells, it is likely that the generation and subsequent analysis of transgenic mice targeting additional components of the ICaL signaling complex will be necessary to understand the physiological process of β-adrenergic regulation of cardiac L-type Ca2+ channels. We thank Angelika Baumgartner for excellent technical assistance.
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