Unchanged β-Adrenergic Stimulation of Cardiac L-type Calcium Channels in Cav1.2 Phosphorylation Site S1928A Mutant Mice*

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 Ca 2ϩ current (I CaL ) by ␤-adrenoceptors, cAMP, and protein kinase A (PKA) 2 (1,2). 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][4][5]. The molecular basis of I CaL regulation by PKA could not be defined conclusively so far (5). This is mainly due to the fact that the extent of regulation of I CaL 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 Ca 2ϩ channels in vivo (2). It is a widely accepted finding that phosphorylation of Ser 1928 of the Ca v 1.2 L-type channel subunit is necessary for ␤-adrenergic regulation of I CaL (5,6).
The amino acid Ser 1928 , which is located in the intracellular C terminus of Ca v 1.2, has been reported to be the only detectable in vivo and in vitro PKA phosphorylation site of the Ca v 1.2 subunit (6 -10). Electrophysiological studies using heterolo-gous expression of the Ca v 1.2 subunit reported contrary findings, i.e. stimulation (11) or no stimulation (12) of I CaL by PKA when only the Ca v 1.2 subunit was expressed. In contrast, the necessity of coexpression and phosphorylation of the Ca v ␤2a subunit was reported (13). Recently, the functional importance of Ca v ␤2a phosphorylation for ␤-adrenergic regulation of I CaL has been questioned (14). Regardless of these findings, mutation of Ca v 1.2 Ser 1928 to alanine prevented phosphorylation and regulation of the channel by PKA in heterologous expression systems (15)(16)(17).
In contrast to these reports, Ganesan et al. (18) postulated that at least 70% of the ␤-adrenergic regulation of I CaL in virally transducted heart cells cannot be attributed to the Ser 1928 phosphorylation event. However, the viral infection system by Ganesan et al. (18) only partially reconstituted the regulation of I CaL (the PKA-mediated increase in current was only 50%).
On the other hand, Hulme et al. (6) recently correlated ␤-adrenergic stimulation with phosphorylation of Ser 1928 and functional up-regulation of I CaL in intact ventricular myocytes. In addition to these findings, Oliveria et al. (17) postulated a critical role for the Ser 1928 phosphorylation in the PKA-mediated regulation of Ca v 1.2 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 Ca v 1.2 S1928A mutation.

EXPERIMENTAL PROCEDURES
Generation of Mice Lacking the Ser 1928 Phosphorylation Site on Ca v 1.2-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 3Ј-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/Svϫ129/ Sv-CP F1) (19). Positive clones were identified by PCR and confirmed by Southern blot using an outer probe (5Ј-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 heterozy-gotes 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 (Ca v 1.2 S1928A-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 l/ml; Sigma), phenylmethylsulfonyl fluoride (200 mmol/liter), calpain inhibitor I (8 g/ml), and calpain inhibitor II (8 g/ml). Hearts from adult mice were frozen and pulverized under liquid N 2 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-Ca v 1.2 and -Ca v ␤2 a antibodies have been described previously (20,21). Antibodies against the catalytic (PKAc) and regulatory subunits (RII␣ and RII␤) of cAMP-dependent protein kinase were purchased from BD Biosciences. The antibodies against GST and against the ␤ 1 -adrenergic receptor were obtained from Calbiochem and Upstate, respectively. The phosphospecific antibody against Ser(P) 1928 was generated by CovalAb against the peptide NH 2 -C-LGRRA(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). 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/5bromo-4-chloro-3-indolyl phosphate as described in Ref. 20. To quantitatively evaluate Ser 1928 phosphorylation, blots were first probed with anti-phospho-Ser 1928 antibody and subsequently with anti-Ca v 1.2 antibody to correct for variability in the amount of total Ca v 1.2. The ratio of the antiphospho-Ser 1928 antibody to the anti-Ca v 1.2 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. Middle, targeting vector. Neo, neomycin resistance gene; TK, thymidine kinase gene with loxP sequence (triangles) at both sides. The S1928A alanine substitution for serine is shown. Bottom, knock-in locus after homologous recombination and Cre-mediated deletion of resistance markers. The 5Ј-probe, which contains genomic sequence outside of targeting vector, is depicted as a solid bar. K, KpnI; N, NotI; B, BamHI; C, ClaI; X, XhoI, P, PstI; kb, kilobases. b, top, sequence analysis of genomic DNA in the region coding for Ser 1928 from Ca v 1.2 S1928A-129B6F2 mice. Bottom, Southern blot using the 5Ј-probe. Wild type (ϩ/ϩ), heterozygote (ϩ/SA), and homozygote (SA/SA) DNA are indicated. c, normal atrial and ventricular expression levels of components of the ␤ 1 -AR/PKA signaling cascade in hearts of Ca v 1.2 S1928A-129B6F2 compared with the wild type as shown by immunoblots. d, Ca v 1.2 S1928A-129B6F2 and wild type mouse ventricular homogenates were subjected to immunoprecipitation (IP) with an anti-Ca v 1.2 antibody, and the immunoprecipitates were analyzed by immunoblotting. Immunoprecipitation of Ca v 1.2 led to coprecipitation of PKAc, RII␣, and RII␤, indicating an association between the proteins.

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Phosphorylation with PKA-For PKA phosphorylation reactions, full-length Ca v 1.2 was immunoprecipitated with the Ca v 1.2-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 MgCl 2 , 0.5 mmol/liter EGTA, 0.5 mmol/liter dithiothreitol). Phosphorylation was carried out by mixing the Sepharose pellets with 33 mol/liter Mg-[␥-32 P]ATP (6.6 Ci/reaction). The reaction, initiated by the addition of 5 units of PKAc (Sigma) to either the GST fusion proteins or immunoprecipitated Ca v 1.2 complexes, was carried out at 23°C for 5 min (GST fusion proteins) or 10 min (immunoprecipitated Ca v 1.2 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 32 P i 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% O 2 , 2% CO 2 .  (3). PKI, reactions in the presence (ϩ) or absence (Ϫ) of 1 mol/liter PKA inhibitor peptide. Anti-GSTantibody was used as loading control. b, GST fusion proteins (wild type fragments amino acids 1509 -1733, amino acids 1733-COOH, and amino acids 1733-COOH S 1928A fragment) were phosphorylated by PKA, size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose, analyzed by autoradiography, and immunoblotted with a phospho-specific antibody recognizing phosphorylated Ser 1928 (pS1928). c, autoradiogram of wild type and S1928A mutant Ca v 1.2 phosphorylated by the catalytic subunit PKAc. Strong phosphorylation was only detectable in the wild type protein, whereas Ca v 1.2 expression levels were unchanged. The graph shows quantification of the autoradiography signals (n ϭ 5) normalized to Ctr levels. *, p Ͻ 0.001 t test. d, absence of phosphorylation at Ser 1928 in phosphomutants. Heart samples from wild type (Ctr) and homozygote (Ki) mice were prepared for immunoblot analysis using antibodies against phosphorylation site Ser 1928 (top lane, left), total Ca v 1.2 (middle), or Ca v ␤ 2 (bottom). Quantification of the immunoblot signals (bar graph, normalized to Ctr levels) shows that phosphorylation is absent in Ca v 1.2 S1928A-129B6F2 mice, whereas Ca v 1.2 expression level is normal. n ϭ 5, *, p Ͻ 0.001 t test. Preincubation with antigenic peptide (top lane, right) abolished the immunoblot signal.
Electrophysiology-Calcium currents (I Ca ) 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 tetraethyl-ammonium⅐Cl, 2 mM MgCl 2 , 1.8 mM CaCl 2 , 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 MgCl 2 , 5 mM MgATP, 10 mM HEPES, and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-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 (Ϫ40 to ϩ80 mV in 10-mV steps) from a holding potential of Ϫ40 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 mol/liter) on I Ca 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 PulseϩPulsefit software version 8.54 (HEKA, Lambrecht, Germany), and data were stored for later off-line analysis. Leak compensation was performed online in PulseϩPulsefit when necessary. All data are expressed as the means Ϯ S.E. Values of p Ͻ 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/O 2 . 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 ϫ 41 ϫ 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 ϫ 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.  (Ctr, top graph) and Ca v 1.2 S1928A-129B6F2 (Ki, bottom graph) ventricular myocytes. b, representative calcium currents elicited by 200-ms test pulses from Ϫ40 to 10 mV before and after exposure to 100 nmol/liter Iso. c, mean current densities of I CaL of wild type (squares) and Ca v 1.2 S1928A-129B6F2 (triangles) ventricular myocytes. DECEMBER 12, 2008 • VOLUME 283 • NUMBER 50

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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).

RESULTS AND DISCUSSION
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 (Ca v 1.2 S1928A-129B6F2 ). The mutants showed no overt cardiovascular phenotype and bred normally. Ca v 1.2 S1928A-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 analy-sis. The point mutation and the loxP site did not alter the expression of the Ca v 1.2 protein (Fig. 1c). The Ca v 1.2 S1928A-129B6F2 mutation did not decrease the expression of ␤ 1 -adrenoceptors or the catalytic and regulatory PKA subunits PKAc, RII␣, and RII␤ in the mutants. Coimmunoprecipitation demonstrated that the macromolecular complex between Ca v 1.2, PKAc, RII␣, and RII␤ (7, 23) was preserved (Fig. 1d). Using GST fusion proteins, we corroborated that mutation of Ser 1928 to alanine in the C terminus of the Ca v 1.2 results in a complete loss of cAMP-mediated phosphorylation in vitro (Fig. 2a).
Next, we developed a phosphospecific antibody to detect Ser 1928 phosphorylation. In a PKA phosphorylation assay of GST fusion proteins, anti-phospho-Ser 1928 antibody immunoreactivity was only detected in the C-terminal fragments containing Ser 1928 (GST-(1733-2171)) and was abolished both by the PKA inhibitor PKI and also when a single substitution (S1928A) was introduced into the substrate sequence (Fig. 2b). These findings highlight that the anti-Ser 1928 antibody only detects Ca v 1.2 phosphorylated at Ser 1928 . To extend these in vitro data to the Ca v 1.2 S1928A-129B6F2 animal model, we next evaluated phosphorylation of cardiac Ca v 1.2 subunits from these mice.
Phosphorylation by PKA in the presence of [␥-32 P]ATP and the catalytic subunit of PKA (5 units) was examined using an immunoprecipitation protocol and autoradiography. Only weak phosphoprotein signals (9.4 Ϯ 4.8% of control level) corresponding to Ca v 1.2 were observed when heart preparations from Ca v 1.2 S1928A-129B6F2 mutants were used, whereas in controls Ca v 1.2 phosphorylation could be readily detected (Fig. 2c). In strong agreement with these results, detection of Ca v 1.2 by the phosphospecific antibody to Ser 1928 was barely detectable in the homozygotes (4.4 Ϯ 2.1% of control level). Specificity of the anti-Ser 1928 antibody in Ca v 1.2 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 Ser 1928 is the only easily detectable Ca v 1.2 PKA phosphorylation site. Moreover, the Ca v 1.2 subunit lacking this phosphorylation site is properly targeted to the Ca v 1.2-associated proteins (7,23), 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 Ca v 1.2 signaling complex.
Next, we looked at whether the lack of this phosphorylation site on Ca v 1.2 affects ␤-adrenergic regulation in adult litter- mate 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 Ca v 1.2 S1928A-129B6F2 mice. To quantitatively correlate phosphorylation of Ser 1928 with regulation of I CaL , we measured ␤-adrenergic stimulation of whole-cell I CaL by isoproterenol (Iso). Iso treatment increased I CaL in control mice 280 Ϯ 25% and in Ca v 1.2 S1928A-129B6F2 mice 268 Ϯ 20%, respectively (Fig. 3,   a and b). In addition to the unchanged Iso-induced increase in peak inward I CaL, the 10-mV left shift in the V1 ⁄ 2 of activation was preserved in cardiomyocytes from Ca v 1.2 S1928A-129B6F2 mice (Fig. 3a).
In agreement with the Iso results, we found that the adenylate cyclase agonist forskolin enhanced I CaL in both control and mutant cardiomyocytes to identical levels (228 Ϯ 39% versus 240 Ϯ 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 I CaL by a G s subunit (24, 25) was not operative.  by  H89. b, no increase in peak I Ca from cardiomyocytes in the presence of 10 M H89 in response to 100 nM isoproterenol. Cells were preincubated for 3-5 min in 10 M H89. c, after 10 min of superfusion with the selective ␤ 1 -adrenoreceptor-antagonist CGP 20712A (100 nM) or the selective ␤ 2 -adrenoreceptorantagonist ICI 118551 (100 nM), cells were superfused with isoproterenol (100 nM) for 5 min. Increases in peak current are indicated. n.s., not significant. FIGURE 6. No changes in ␤-adrenoreceptor-dependent modulation of heart rate and hemodynamic function in Ca v 1. 2 S1928A mutant mice in vivo. a, normal sympathetic up-regulation of heart rate in mice in response to application of isoproterenol (Iso, 0.1 mg/kg body weight). Shown is the time course of heart rate 15 min before and 45 min after intraperitoneal injection of Iso. Drugs were injected at t ϭ 15 (arrow). Isoproterenol increased the heart rate of Ctr (open circles, n ϭ 5) and Ca v 1.2 S1928A animals (filled circles, n ϭ 6) from 600 to ϳ730 beats per min. b, representative M mode echocardiography after isoproterenol infusion (0.5 mg/kg body weight) of a control (Ctr, left image) and a Ca v 1.2 S1928A animal. (Ki, right image). c, left ventricular FS assessed by echocardiography in anesthetized mice before and after intraperitoneal isoproterenol injection (0.5 mg/kg body weight). The increase of FS was not significant different between the genotypes.
To further substantiate these findings, we characterized the functional role of additional components of the ␤-adrenergic pathway in I CaL regulation. The PKA inhibitor H89 consistently blocked the isoproterenol-induced increase in I CaL in littermatched control and Ca v 1.2 S1928A-129B6F2 cardiomyocytes (Fig. 5,  a and b).
The isoproterenol effect was also completely suppressed by application of 0.1 M of the ␤ 1 -adrenergic blocker CGP20712A and partially by 0.1 M of the ␤ 2 -adrenergic blocker ICI118551 in wild type and mutant mice (Fig. 5c). Taken together, these data rule out a switch of the physiological ␤-adrenergic regulation of cardiac Ca v 1.2 in Ca v 1.2 S1928A-129B6F2 mice.
The results shown so far did not rule out the possibility that phosphorylation of Ser 1928 is required for the in vivo regulation of cardiac function. First, we looked at a potential role of Ca v 1.2 Ser 1928 phosphorylation in the regulation of heart rate and rhythm using ECG telemetry. There were no detectable differences in basal heart rate (587 Ϯ 24 beats/ min versus 567 Ϯ 27 beats/min, t test, p Ͼ 0.05). Isoproterenol infusion increased heart rate to the same level (734 Ϯ 15 beats/min versus 741 Ϯ 6 beats/min, t test, p Ͼ 0.05) in both control and Ca v 1.2 S1928A-129B6F2 animals (Fig. 6a).
We next tested the consequence of ablation of Ca v 1.2 Ser 1928 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 ␤-adrenergic stimulation in vivo is not dependent on Ser 1928 .
Taken together, our results support the notion that PKA phosphorylation of Ser 1928 of Ca v 1.2 is functionally not involved in ␤-adrenergic regulation of I CaL in murine ventricular cardiomyocytes. The results are in line with the previous finding that ␤-adrenergic stimulation requires a PKAmediated phosphorylation step (1,6,9,11,13,16,18,24). Many questions remain to understand the ␤-adrenergic regulation of Ca 2ϩ channels. What is the physiological substrate of PKA in the Ca v 1.2 channel complex responsible for acute stimulation of I CaL, if not Ser 1928 ? The phosphorylation target could be the Ca v ␤ 2a subunit (13) or the giant protein AHNAK (26). Our studies provide the basis to address these critical questions in the future. Because it has been difficult to reconstitute reproducibly I CaL regulation in cultured cells, it is likely that the generation and subsequent analysis of transgenic mice targeting additional components of the I CaL signaling complex will be necessary to understand the physiological process of ␤-adrenergic regulation of cardiac L-type Ca 2ϩ channels.