Deletion of the C-terminal Phosphorylation Sites in the Cardiac β-Subunit Does Not Affect the Basic β-Adrenergic Response of the Heart and the Cav1.2 Channel*

Background: β-Adrenergic receptors stimulate cardiac ICa via PKA-dependent phosphorylation. Results: Deletion of the C-terminal phosphorylation sites in the β2 gene did not affect isoproterenol-stimulated ICa. Conclusion: Phosphorylation of the C terminus of the β2 subunit in vivo does not contribute to β-adrenergic regulation of ICa. Significance: The PKA-dependent regulation of ICa does not require the C terminus of the β2 subunit. Phosphorylation of the cardiac β subunit (Cavβ2) of the Cav1.2 L-type Ca2+ channel complex has been proposed as a mechanism for regulation of L-type Ca2+ channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Cavβ2 gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; βStop mouse). This mutation prevented translation of the Cavβ2 C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac βStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type ICa. The regulation of the L-type current by stimulation of the β-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). βStop mice were cross-bred with mice expressing the Cav1.2 gene containing the mutation S1928A (SAβStop) or S1512A and S1570A (SFβStop) in the C terminus of the α1C subunit. The β-adrenergic regulation of the cardiac ICa was unaltered in these mouse lines. In contrast, truncation of the Cav1.2 at Asp1904 abolished β-adrenergic up-regulation of ICa in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Cavβ2, Ser1928, Ser1512, and Ser1570 of the Cav1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Cav1.2 channel.

The cardiac L-type Ca 2ϩ current (I Ca ) is regulated by a number of protein kinases including PKA, CaMKII, 2 PKC, Akt/PKB, and PKG (1,2). Regulation of the cardiac Ca v 1.2 channel by ␤-adrenoceptors, cAMP, and PKA has been implicated as basic mechanism of the fight or flight reaction of an animal (3,4). Phosphorylation of some channel subunits plays a critical role in several physiological cardiac processes, e.g. excitation-contraction coupling, the regulation of positive inotropy and chronotropy, as well as pathological processes such as heart failure (for review, see 2,5). The molecular basis of I Ca regulation by protein kinases could not be defined conclusively so far because expression studies suggested phosphorylation sites on the ␣ 1 subunit and the ␤ 2 subunit of the L-type calcium channel. Phosphorylation sites on the ␣ 1 subunit were invoked for PKA (6 -8), CaMKII (9 -11), PKG (12,13), Akt/PKB (14,15), and by PKC (16 -19). In addition, phosphorylation sites in the Ca v ␤ 2 subunit were reported for PKA (20) at Ser 479/480 (rabbit protein sequence (rbs)) (21), CaMKII (22) at Thr 500 (rbs), PKG (12) at Ser 496 (rbs), and Akt/PKB (14,15,23,24) at Ser 576 (rbs). The amino acids modified by protein kinases in Ca v ␤ 2 or Ca v 1.2 in the protein sequence from rabbit, rat, and mouse are listed in supplemental Table 1.
This very impressive work of several groups missed a clear statement, if the phosphorylation of one subunit was necessary to regulate I Ca in vivo. Previously, we investigated whether phosphorylation of Ser 1928 of the ␣ 1 subunit was a necessary step for the ␤-adrenergic regulation of the cardiac I Ca in vivo. Mutation of the Ser to Ala did not affect the ␤-adrenergic regulation (25), raising the possibility that phosphorylation of the Ca v ␤ 2 subunit by PKA (20) might be the requested regulatory step. Therefore, we generated a mouse line that contained a stop codon in exon 14 of the mouse Cacnb2 gene after Pro 501 (␤Stop). This stop codon resulted in a truncated ␤ 2 subunit protein that lacked the potential phosphorylation sites for PKA, PKG, Akt/PKB, and CaMKII. Basal properties of the Ca v 1.2 current were unaffected as expected from the report that deletion of the Cacnb2 gene in the adult heart has minimal effects on I Ca (26). We crossed the ␤Stop line with the S1928A (25) and SF (S1512A and S1570A) (27) mouse lines that contain well characterized phosphorylation sites for PKA and CaMKII, respectively. Again, the basal properties of the Ca v 1.2 current were unaffected, suggesting that ␤-adrenergic regulation of the Ca v 1.2 channel may be mediated by other phosphorylation sites, e.g. Ser 1700 of the ␣ 1 subunit (8).

EXPERIMENTAL PROCEDURES
All substances used were of the highest purity available. Amino acid numbering is according to the Mus musculus Cacnb2 sequence (GenBank accession number Q8CC27) or to the Oryctolagus cuniculus (rabbit) Cacnb2 sequence (GenBank accession number X64297). The amino acids modified by protein kinases in Ca v ␤ 2 or Ca v 1.2 in the protein sequence from rabbit, rat, and mouse are listed in supplemental Table 1. Within this paper we refer to the amino acid modified in the rabbit sequence of GenBank, X64297.
Generation of Mice Lacking the C Terminus of Ca v ␤ 2 -To construct the targeting vector, a 7.3-kb fragment containing exons 13-14 of CACNB2 was isolated from 129/Sv mouse genomic DNA. The targeting vector included a 1.6-kb short arm and 5.7-kb long arm with PGK-neo and the thymidine kinase gene (tk) flanked by two loxP sites. The 3Ј-side long arm contained exon 14 with the stop codon TGA in-frame after Pro 501 and the phosphorylation sites Ser 529/530 (corresponding to Ser 479/480 rbs), Ser 545 (corresponding to Ser 496 rbs), Thr 549 (corresponding to Thr 498 rbs), and Ser 625 (corresponding to Ser 574 rbs) behind the stop codon (see supplemental Table 1). All mutation procedures were carried out by QuikChange II sitedirected mutagenesis (Stratagene). The targeting construct was electroporated into R1 ES cells (129/Svϫ129/Sv-CP F1). Positive clones were identified by PCR and confirmed by Southern blotting using a probe on the neo gene. One positive clone was detected and injected in C57BL/6 blastocysts. Chimeras were crossed to C57BL/6 mice. By crossing with a Cre-recombinase expressing transgenic B6.C-Tg (CMV-cre)1Cgn/J mouse strain, the neo tk marker genes were excised. 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 (75:131:64). For all analyses, filial generation 2 (F2) mice with 129/Sv and C57BL/6 hybrid genetic background were used. All procedures relating to animal care and treatment were authorized by the "Regierung von Oberbayern" and conformed to the institutional, governmental, Directive 2010/ 63/EU of the European Parliament guidelines and to the Care and Use of Laboratory Animals published by the US National Institutes of Health. Anesthetized mice (1.5% isoflurane) were euthanized by cervical dislocation.
Membrane Preparation and Immunoblotting-Frozen heart and brain tissue were pestled to a fine powder and homogenized in membrane preparation buffer (20 mM EDTA, 20 mM EGTA, 10 mM Tris, 300 mM NaCl, pH 7.4, inhibitors per ml buffer: 8 g of calpain inhibtor I (Roche Applied Science), 8 g of calpain inhibitor II (Roche Applied Science), 1 l of phenylmethylsulfonyl fluoride (PMSF; Fluka), and 2 l of protease inhibitor mixture (Sigma)). Cell organelles were separated by centrifugation, the supernatant containing the membrane proteins was centrifuged at 100,000 ϫ g for 30 min, and the pellet was solubilized in deoxycholate buffer (20 mM EDTA, 10 mM EGTA, 10 mM Tris-HCl, pH 7.4), 1% deoxycholate) for 20 min. Membrane proteins were separated by centrifugation at 100,000 ϫ g for 30 min. The supernatant was aliquoted and stored at Ϫ80°C, and protein concentration was measured according to the BCA method (Pierce). 50 g of protein were separated per lane on 10% SDS-polyacrylamide gels, blotted, and probed with antibodies by using a chemiluminescence detection system.
Electrophysiological Recordings-Whole cell I Ca was measured at room temperature from rod-shaped, striated, calciumtolerant myocytes within 1-24 h after isolation. Stimulation and data acquisition were performed as described in Refs. 27, 30, 31. Facilitation of I Ca was measured during a triple pulse protocol with a 200-ms control pulse to 0 mV (V1) followed by a 200-ms prepulse (V pre ) to ϩ80 mV followed by 200-ms test pulse to 0 mV (V2) (27). The extent of voltage-dependent facilitation was calculated as the ratio of the peak current during V2 and V1. Time constants of I Ca inactivation were obtained by a fit from peak current to the current value at the end of the voltage pulse by a two-exponential function using pClamp 9. Facilitation of I Ca was measured as described in Ref. 30. The stimulatory effect of isoproterenol (100 nmol/liter containing an equal concentration of ascorbic acid) on I Ca was examined after establishing a solid base line. Stimulation of I Ca by isoproterenol was measured at a membrane potential of Ϯ0 mV and is given as percentage of control ( ϭ 100%) determined before superfusion with isoproterenol. All fits showed a correlation coefficient Ͼ0.98.
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 (0.1 mg/kg mouse; Sigma) or phenylephrine (3 mg/kg mouse; Sigma) was dissolved in 0.9% NaCl. After 15 min of base-line recording, the mice were injected intraperitoneally with the drugs. 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.
Echocardiography-Images were obtained using a Vevo 770 Visual Sonics scanner equipped with a 30-MHz probe (Visual Sonics Inc., Toronto, ON, Canada). The mice were lightly anesthetized (1.5% isoflurane) and 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, the diameter at the end of systole minus the diameter at the end of diastole divided by the diameter at the end-diastole) was assessed from the M mode of the parasternal short axis view. Control and mice carrying the various mutations were studied before and after administration of isoproterenol (0.1 mg/kg mouse intraperitoneally).
Statistics-Data are presented as mean Ϯ S.E. Statistical significance was tested by using a (two-tailed) unpaired Student's t test. The null hypothesis was rejected if p Ͻ 0.05.

RESULTS
We report the generation of a mouse line in which the ␤ 2 subunit of the Ca v 1.2 channel complex (Ca v ␤ 2 ) was truncated at Pro 501 (␤Stop mice). For this purpose, we used a gene-targeting strategy that utilized a replacement vector containing a stop codon after proline 501 in exon 14 and a neo tk gene cassette flanked by loxP sites (Fig. 1A). The Ca v ␤ 2 C terminus was truncated to prevent the expression of several putative phosphory-lation sites (PKA Ser 479/480 rbs, PKG Ser 496 rbs, CaMKII Thr 498 rbs, and Akt/PKB Ser 574 rbs; see supplemental Table 1) and to test the physiological relevance of these sites (Fig. 1B). All homozygous ␤Stop mutants analyzed were chimeric F2 mice (mixed sv129 and C57BL/6 background). ␤Stop mice were compared with litter-matched control mice (Ctr ␤Stop). Nomenclature and genotype of mouse lines are outlined in supplemental Table 2. The correct genomic localization (supplemental Fig. 1) and mutation (Fig. 1C) in ␤Stop mice was confirmed by Southern blotting and genomic sequencing. ␤Stop mice were viable, fertile, and reproduced in a 1:2:1 Mendelian ratio (WT 27.7%, heterozygous Ctr ␤Stop 48.5%, ␤Stop 23.7%) (Fig. 1D). Western blot analysis of heart and brain membrane fraction showed no alterations in Ca v 1.2 expression. The expression of the C-terminal truncated Ca v ␤ 2 protein was con- firmed by the C-terminal binding Ca v ␤ 2v2 antibody and the N-terminal binding antibody Ca v ␤ 2 -N4/1195. The C-terminal binding Ca v ␤ 2v2 antibody detected the 75-kDa WT Ca v ␤ 2 protein, but not the truncated Ca v ␤ 2 protein (Fig. 1E), whereas the N-terminal binding Ca v ␤ 2 -N4/1195 antibody detected both the truncated Ca v ␤ 2 protein (58 kDa) and the WT protein (75 kDa) (Fig. 1F). These results show that the ␤Stop mouse expressed the truncated Ca v ␤ 2 protein that missed the reported phosphorylation sites.
Telemetric ECG measurement of heart rate (HR) and activity revealed no differences in WT and ␤Stop mice (supplemental Fig. 2). Both genotypes showed a typical cardiac response to isoproterenol and phenylephrine administration with an increase and a drop in HR, respectively ( Fig. 2A). FS was identical in Ctr ␤stop and ␤Stop mice (Fig. 2B). Isoproterenol doubled FS in both genotypes. These data indicate that the putative PKA phosphorylation sites Ser 479/480 rbs of the Ca v ␤ 2 subunit are not necessary to observe the positive inotropic, ␤-adrenergic regulation of the heart muscle.
To further support the insignificant effect of the Ca v ␤ 2 truncation for cardiac ␤-adrenergic regulation, patch clamp experiments were carried out on isolated cardiomyocytes (CMs). Isolated CMs of either genotype had normal size (WT, 161. It is widely accepted that calcium-dependent facilitation (CDF) is caused by activation of CaMKII followed by phosphorylation of a component of the Ca v 1.2 channel complex. Recently, we reported that CDF requires phosphorylation of Ca v 1.2 at Ser 1512/1570 (27). In contrast, Colbran and co-workers (22) reported that phosphorylation of Ca v ␤ 2 by CaMKII at Thr 500 modulated CDF. Because Thr 500 was not present anymore in the ␤Stop protein, we tested whether or not the truncation of the Ca v ␤ 2 C terminus might affect I Ca facilitation. We compared prepulse facilitation of I Ca in CMs of both genotypes. CDF was not affected by the truncation of the Ca v ␤ 2 protein (Fig. 2F), suggesting that phosphorylation of the Ca v ␤ 2 subunit is not a necessary prerequisite to induce CDF under basal conditions.
In agreement with the ECG results, isoproterenol stimulated I Ca of Ctr ␤Stop and ␤Stop CMs to the same level (Fig. 2G). Representative current traces for a ␤Stop CM are shown in Fig.  2H. Isoproterenol treatment increased I Ca in Ctr ␤Stop CMs by 193 Ϯ 25% and in ␤Stop CMs by 180 Ϯ 24% (Fig. 2G). Furthermore, there was no change in the slow or fast component of inactivation either with or without isoproterenol stimulation (supplemental Table 3). The fast component of inactivation describes Ca 2ϩ -dependent inactivation (CDI), the slow component the voltage-dependent inactivation. Neither inactivation pathway is affected by the C-terminal truncation of the Ca v ␤ 2 protein.
These negative experiments raised the possibility that the positive inotropic effect was mediated by phosphorylation of both the Ca v 1.2 and Ca v ␤ 2 subunit. We tested this hypothesis by cross-breeding the ␤Stop line with the Ca v 1.2 SA or the Ca v 1.2 SF lines. The Ca v 1.2 SA mouse line expresses a Ca v 1.2 channel containing the mutation S1928A (25). Mice homozygous for the double mutation Ca v 1.2 S1928A /Ca v 1.2 S1928A , (SA␤Stop) had the same size and weight as the heterozygous litters. Diurnal cardiac rhythm was not altered in these mice (supplemental Fig. 3). The cell capacitance of Ctr SA␤Stop and double knock-in SA␤Stop CMs was the same (Ctr SA␤Stop: 168.6 Ϯ 12 pF (n ϭ 15); SA␤Stop: 163.0 Ϯ 18 pF (n ϭ 6)). Inactivation time constants of I Ca were not affected by this double mutation (supplemental Table 3). We did not observe an effect of the double mutation on CDF (Fig. 3A). Isoproterenol stimulated FS (Fig. 3B) and HR (supplemental Fig. 4) in both mouse lines to the same extent. No statistically significant difference was noted between the curves. Phenylephrine decreased the HR to the same extent in both genotypes (supplemental Fig. 4). Stimulation of the corresponding CMs by 100 nM isoproterenol increased I Ca by 194.3 Ϯ 19.2% (n ϭ 6) and 205.3 Ϯ 14% (n ϭ 6) in heterozygous Ctr SA␤Stop and homozygous SA␤Stop, respectively (Fig. 3, C  and D).
In the next series of experiments we tested the double mutation Ca v 1.2 S1512/1570A /Ca v 1.2 S1512/1570A , P501Stop (SF␤Stop). Mice homozygous for the double mutation SF␤Stop had the same size and weight as the heterozygous litters. Diurnal cardiac rhythm was not altered in these mice (supplemental Fig. 3). The cell capacitance of Ctr SF␤Stop and double knock-in SF␤Stop CMs was the same (Ctr SF␤Stop: 213 Ϯ 13 pF (n ϭ 9); SF␤Stop: 195 Ϯ 17 pF (n ϭ 8)). Inactivation time constants of I Ca were not affected by this double mutation (supplemental Table 3). As shown for the SF mice (27), CDF was also significantly decreased in the SF␤Stop mice (Fig. 4A).
Isoproterenol stimulated FS (Fig. 4B) and HR (supplemental Fig. 5) in both mouse lines to the same extent. Phenylephrine decreased the HR to the same extent in both genotypes (supplemental Fig. 5). Stimulation of these CMs by 100 nM isoproterenol increased I Ca by 187 Ϯ 17% (n ϭ 10) and 196 Ϯ 26% (n ϭ 8) in the heterozygous Ctr SF␤Stop and homozygous SF␤Stop line, respectively (Fig. 4, C and D). We concluded 3 These results indicated to us that there is no gene dose effect through the deletion of the C terminus of one ␤ 2 gene. Almost identical results have been reported by Meissner et al. (26), which reported the inactivation of both ␤ 2 alleles. Comparison of WT and heterozygous animals should allow the detection of a gene dose effect more easily. However, if no different phenotype has been found between the WT and heterozygous animals, it is generally requested that the heterozygous litter-matched animals are the correct controls to the knockout mice because they carry one WT chromosome and one chromosome carrying the mutated gene. Based on these generally accepted ␤Stop considerations, we tested against the heterozygous, litter-matched CTR animals. Furthermore, heart-specific inactivation of the ␤ 2 subunit gene in adult mice yielded minimal or no effect on I Ca kinetics (see Ref. 26, Figs. 4 and 5).
from this analysis that neither double mutation affected the ␤-adrenergic stimulation of FS in the intact mouse nor that of I Ca in the CMs.
The results presented so far suggested that none of the mutated potential PKA or CaMKII phosphorylation sites was necessary for ␤-adrenergic stimulation of the cardiac I Ca . PKA   needs to bind to the L-type channel complex through a PKAanchoring protein (AKAP) before it can phosphorylate the necessary subunit. The CMs contain several AKAPs that may be an essential part of the ␤-adrenergic regulation (32). Disruption of AKAP5 interfered with ␤-adrenergic-stimulated intracellular Ca 2ϩ transients but not with I Ca (33). AKAPs bind to the C terminus of Ca v 1.2 between amino acids 2026 and 2085. This sequence was not modified in the mouse lines studied, suggesting that PKA was still targeted to the ␤-adrenergic-regulated site. Truncation of the Ca v 1.2 sequence at Asp 1904 (31) or at Gly 1796 (34) leads to a channel that does not bind any more AKAPs. In contrast to a previous in vitro study (20) but in agreement with Fu et al. (34), I Ca of embryonic Ca v 1.2Stop CMs is not stimulated anymore by isoproterenol (Fig. 5, A and C) or forskolin (Fig. 5, B and D), suggesting that the amino acids C-terminal to Asp 1904 are essential for the adrenergic up-regulation of I Ca in the heart.

DISCUSSION
Adrenergic up-regulation of the cardiac I Ca is an extensively studied physiological phenomenon that was recognized in the seventies (35) to be regulated by cAMP. Since then evidence has been published that ␤-adrenergic stimulation requires a PKAmediated phosphorylation step (3, 4, 7, 20, 36 -39). However, the molecular mechanism of ␤-adrenergic regulation of Ca v 1.2 channel remains unsolved. Previously, it was found that the mutation S1928A of the Ca v 1.2 protein did not abolish ␤-adrenergic regulation of the heart and I Ca (25), supporting the notion that phosphorylation of S1928 by PKA was not an obligatory step to allow ␤-adrenergic regulation of the murine heart.
The Ca v ␤ 2 subunit has been promoted as an alternative substrate for PKA (20,40,41). Initially, it was suggested that PKA-dependent up-regulation of the expressed I Ca requires truncation of the Ca v 1.2 protein at amino acid 1905 and the co-expression of the Ca v ␤ 2 subunit (20). Truncation of the murine Ca v 1.2 channel at Asp 1904 resulted in death around birth (31) and the inability of isoproterenol to stimulate the truncated channel (Fig. 5). Similar results have been reported, when the Ca v 1.2 protein was truncated at Gly 1796 (34). These negative results are most likely caused by the deletion of the AKAP binding sequence (32). AKAPs are components that target various proteins of the ␤-adrenergic signaling cascade to Ca v 1.2 (38). These results clearly demonstrate that truncation of the Ca v 1.2 protein in vivo is not required for the adrenergic regulation.
Truncation of the Ca v ␤ 2 subunit at P501 by site-directed mutagenesis removed the "classical" PKA phosphorylation sites and that for PKG, CaMKII, and PKB. Removing these reported phosphorylation sites had no effect on the basic properties of the murine cardiac I Ca . The Ca v ␤ 2 Stop mice showed normal ␤-adrenergic regulation, CDI, CDF, and basic behavior. From these results we conclude that these reported phosphorylation sites are not necessary for the basic regulation of the channel by PKA, CaMKII, PKG, and PKB.
The reported results do not rule out the possibility that PKA modified an additional site on the truncated Ca v ␤ 2 subunit that was necessary for ␤-adrenergic regulation of the channel (42). This consideration appears unlikely in view of the report that deletion of the Ca v ␤ 2 subunit in the adult heart does not result in an severe phenotype (26). The negative results reported here are only relevant for the relative classical tests carried out in this study. It could be that removal of these phosphorylation sites may alter more subtle cardiac functions that have not been associated so far with a phosphorylation event at the Ca v ␤ 2 subunit.
An alternative possibility is that PKA phosphorylates simultaneously sites at the Ca v ␤ 2 and the Ca v 1.2 subunit. This possibility was tested with two additional mouse lines. However, the combination of the Ca v ␤ 2 mutation with mutation at the C terminus of the Ca v 1.2 protein did not influence the cardiac response to ␤-adrenergic stimulation. However, the Ca v ␤ 2 mutation did not affect the reduced CDF caused by the SF mutation (27). Our results do not rule out the possibility that phosphorylation at these sites might affect parameter of the Ca v 1.2 channel that have not been studied under our condition. However, the physiological significance of these putative parameters appears to be restricted because deletion of the cardiac Ca v ␤ 2 gene in the adult mouse caused only negligible changes in the cardiac performance (26).
An alternative target for PKA and ␤-adrenergic regulation of the heart has been proposed recently (8). Expression studies implicated PKA-dependent phosphorylation of Ser 1700 and Thr 1704 in the C terminus of Ca v 1.2 to be important for the ␤-adrenergic regulation (8). The phosphorylation needs to be combined with proteolytic cleavage of the mature Ca v 1.2 protein close to amino acid 1800. The cleaved distal C terminus has to stay with the truncated Ca v 1.2 channel to allow ␤-adrenergic regulation. The distal part inhibits I Ca of the truncated Ca v 1.2 channel. The inhibition is then removed by ␤-adrenergic stimulation (8).
For ␤-adrenergic regulation of the expressed I Ca , an additional function of the distal part, its AKAP binding site, is required. The AKAP binding site allows the close positioning of PKA to the Ca v 1.2 channel. Partial verification of the AKAP concept has been given by Nichols et al. (33), Fuller et al. (8) and by the results of this report. The I Ca of embryonic CMs expressing a truncated Ca v 1.2 channel (see Fig. 7 in Ref. 34) was not stimulated by isoproterenol or forskolin. To support further the above concept, in vivo mutation of Ser 1700 /Thr 1704 is necessary to show that ␤-adrenergic regulation of I Ca requires the phosphorylation of Ser 1700 /Thr 1704 .
The reported results suggest that the C-terminal phosphorylation sites of the Ca v ␤ 2 subunit are not used to regulate basic properties of the murine cardiac I Ca . In contrast, CaMKII-dependent phosphorylation of the C terminus of Ca v 1.2 is necessary for CDF. The results support again the previous notion (31,34) that the distal C terminus of the Ca v 1.2 channel is necessary for ␤-adrenergic regulation of murine I Ca .