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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Oct 19;103(44):16574–16579. doi: 10.1073/pnas.0607294103

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac CaV1.2 channels during β1-adrenergic regulation

Joanne T Hulme 1, Ruth E Westenbroek 1, Todd Scheuer 1, William A Catterall 1,*
PMCID: PMC1637623  PMID: 17053072

Abstract

During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by CaV1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of β-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the α1 subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to β-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective β1- and β2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N′,N′-tetraacetic acid (BAPTA)–acetoxymethyl ester to buffer intracellular calcium results in only β1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to β1-adrenergic receptors. Thus, β-adrenergic stimulation results in phosphorylation of S1928 on the CaV1.2 α1 subunit in intact ventricular myocytes via both β1- and β2-adrenergic receptor pathways, but the β2-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. Our results correlate phosphorylation of S1928 with β1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.

Keywords: calcium channel, cAMP, heart, protein kinase, sympathetic regulation

The primary calcium current in cardiac myocytes is conducted by Cav1.2a channels (1, 2). Calcium entering through these channels triggers release of calcium from the sarcoplasmic reticulum, which in turn activates contraction. Modulation of calcium entry through the CaV1.2 channel is a key point of regulation of cardiac contraction by the sympathetic nervous system, primarily through epinephrine and norepinephrine acting at β-adrenergic receptors (1, 3). Activation of β-adrenergic signaling leads to 3- to 4-fold increases in calcium current (2), mediated by protein kinase A (PKA) phosphorylation (1–3). Increases in calcium current are produced by activation of adenylyl cyclase with forskolin, inhibition of phosphodiesterases, and activation of PKA with cyclic nucleotide analogs (4–6).

Cardiac calcium channels are heteromeric protein complexes (7). The pore-forming α1 subunit consists of four homologous domains surrounding an ion conduction pore and contains the gating machinery and the receptor sites for calcium antagonist drugs (8). It is associated with disulfide-linked α2 and δ subunits and an intracellular β subunit. The full-length α1 subunit is 242 kDa, but it is proteolytically processed to 210 kDa in vivo (9, 10). The proteolytically processed distal C terminus remains noncovalently associated with the truncated α1 subunit and exerts potent autoinhibitory effects on channel function (11, 12). Biochemical studies showed that the cleaved α1 subunit lacking its distal C-terminal tail is not a substrate for phosphorylation by PKA (13–15), whereas the full-length α subunit was readily phosphorylated (16) on serine 1928 (S1928) in the distal C-terminal domain (9, 17). Anchoring of PKA to the proteolytically processed distal C-terminal domain by an A-kinase anchoring protein (AKAP15) is required for effective regulation of CaV1.2 channels in ventricular myocytes (18). In addition, the β2a subunit is phosphorylated by PKA (19, 20) at S459 and S478/479 (21), and this phosphorylation may be important for regulation (22).

In this study, we measured phosphorylation of S1928 in intact cardiac myocytes by using a phosphospecific antibody and compared the level of phosphorylation and the extent of stimulation of calcium currents by the β1- and β2-adrenergic pathways. Our results show that S1928 is phosphorylated in response to activation of both the β1- and β2-adrenergic receptor pathways in physiological solution, but the β2 response is prevented by buffering intracellular calcium. When intracellular calcium transients are prevented, only the β1-adrenergic pathway leads to substantial increase in whole-cell calcium current, and S1928 is phosphorylated concurrently with this regulation.

Results

The specificity of the phosphospecific antibody anti-CH3P against phosphorylated S1928 was probed by immunoblot analysis of Cav1.2a channels transiently expressed in tsA-201 cells. Anti-CH3P recognized a band of ≈250 kDa corresponding to the Cav1.2a channel in cells treated with 10 μM forskolin to activate PKA and 100 nM okadaic acid to inhibit phosphoprotein phosphatases (Fig. 1A, WT), but no immunoreactivity was observed in untreated cells. No phosphoprotein band was detected in cells expressing S1928A mutant channels (Fig. 1A, S1928A), even though channel expression was equivalent (Fig. 1A Lower). These results indicate that only phosphorylated S1928 on the Cav1.2a channel is recognized by anti-CH3P.

Fig. 1.

Fig. 1.

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Phosphorylation of S1928 in the α1 subunit of CaV1.2 channels in transfected tsA-201 cells. (A) Recombinant WT and S1928A CaV1.2a channels were expressed in tsA-201 cells. Cells were exposed to forskolin (10 μM) plus okadaic acid (OA; 100 nM) for 10 min. Solubilized protein extracts were separated by SDS/PAGE, and immunoblots were probed with anti-CH3P (Upper) or anti-CNC1 (Lower). (B) Confocal images of the center of tsA-201 cells expressing WT and S1928A CaV1.2a channels labeled with anti-CH3P before (Control) and after (+Forskolin/OA) a 10-min stimulation with 10 μM forskolin plus 100 nM OA. (Scale bar: 20 μm.)

In immunocytochemical experiments (Fig. 1B), anti-CH3P produced only background staining of untreated cells expressing WT Cav1.2a channels (Fig. 1B Upper Left), but intense staining was observed in cells pretreated with forskolin and okadaic acid (Fig. 1B Upper Right). This increased staining intensity was not observed in cells expressing S1928A channels (Fig. 1B Lower), demonstrating specific recognition of Cav1.2a channels phosphorylated at S1928 by anti-CH3P.

We examined phosphorylation of S1928 in response to isoproterenol (Iso, 100 nM) stimulation of acutely isolated rat ventricular myocytes, by using anti-CH3P to recognize phosphorylated S1928 and anti-CH2 against the distal C-terminal tail of Cav1.2a to recognize total Cav1.2 channel protein. Staining with anti-CH2 produced a striated pattern, consistent with staining of the junctions between the transverse (t−) tubules and sarcoplasmic reticulum (Fig. 2 A and B), where Cav1.2a channels are located in high density (23–25). Staining intensity was similar across the width of the myocyte and was unchanged by treatment with Iso (Fig. 2 A and B). When Iso-treated cells were stained with anti-CH3P, a dramatic increase in immunofluorescence was observed throughout the cell (Fig. 2E), indicating strong phosphorylation of S1928 in intact cardiac myocytes.

Fig. 2.

Fig. 2.

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Iso-induced increases in phosphorylation of S1928 in the distal C-terminal tail of CaV1.2a channels in isolated rat ventricular myocytes. (A–C) Confocal (A and B) and mean line scan (C) images of a central plane of rat ventricular myocytes labeled with anti-CH2 before (A) and after (B) stimulation with 100 nM Iso. (D–F) Confocal (D and E) and mean line scan (F) images of myocytes labeled with anti-CH3P before (D) and after (E) stimulation with 100 nM Iso. The gain and offset of the microscope were kept constant for each experiment. (Scale bar: 10 μm.)

Visual examination of the distribution of immunocytochemical staining can be misleading. To provide a more quantitative measurement, we used line scans of confocal optical sections across the cells, taken in a fluorescence range where stain intensity was approximately linear (see Methods). This analysis confirmed that there was no change in stain intensity between control and Iso-treated cells for Cav1.2a channel protein recognized by anti-CH2 (Fig. 2C). In contrast, there was a large increase in stain intensity for Cav1.2a channels containing phosphorylated S1928 at the cell surface and across the width of the cells (Fig. 2F).

S1928 is phosphorylated by PKA in vitro (9, 17). To determine whether this site was phosphorylated by stimulation of PKA in vivo, forskolin was used to activate adenylyl cyclase. This treatment resulted in an increase in phosphorylation of S1928 that was similar to that produced by Iso (Fig. 3).

Fig. 3.

Fig. 3.

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Increased phosphorylation of S1928 with forskolin. Myocytes were labeled with anti-CH3P in control or after a 10-min stimulation with 10 μM forskolin. (A) Representative confocal images of a central plane of control (Lower) and forskolin-treated (Upper) myocytes. (B) Mean line scan images. (Scale bar: 10 μm.)

Iso is a nonselective activator of all β-adrenergic receptors. We used specific antagonists of β1 and β2 receptors to quantify phosphorylation of S1928 mediated by stimulation of the β1- or β2-adrenergic pathways (Fig. 4). Treatment of the cells with the β1-adrenergic antagonist CGP 20712A before treatment with Iso reduced the fluorescence by 79% (Fig. 4 B and M) relative to cells treated with Iso alone (Fig. 4 A and M). On the other hand, pretreatment with the β2-adrenergic receptor antagonist ICI 118551 caused a 42% reduction in the response to Iso (Fig. 4 C and M). Application of both inhibitors blocked all response to Iso (Fig. 4 D and M). Treatment of unstimulated cells with CGP 20712A (Fig. 4F) had little effect on phosphorylation, but pretreatment with ICI 118551 (Fig. 4G) or both antagonists (Fig. 4H) reduced the control level of phosphorylation, presumably by blocking constitutive activity of β2-adrenergic receptors (26, 27). The fluorescence blocked by the β1 antagonist CGP 20712A and the β2 antagonist ICI 118551 had similar distributions across the cell profile (Fig. 4N). The superadditivity of the inhibition by the two inhibitors (121%) likely reflects a combination of remaining nonlinearity in our measurements of fluorescence, despite our efforts to work in the linear range (see Methods), plus the effect of inhibiting the basal activity of β2 receptors with ICI 118551.

Fig. 4.

Fig. 4.

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Effect of CGP 20712A and ICI 118551 on the Iso-induced increase in S1928 phosphorylation. (A–O) Confocal (A–L) and mean line scan (M–O) images of central planes of rat ventricular myocytes labeled with anti-CH3P. For β-adrenergic receptor inhibition experiments, myocytes were incubated with CGP 20712A (1 μM; B, F, and J), ICI 118551 (1 μM; C, G, and K), or CGP 20712A plus ICI 118551 (1 μM; D, H, and L) for 20 min at 37°C. Cells were then exposed to Iso (100 nM, A–D), control extracellular bath solution (E–H), or Iso (100 nM) plus BAPTA-AM (5 μM) in extracellular bath solution (I–L) for 10 min. Iso, red; ICI 118551, green; CGP 20712A, blue; ICI118551 plus CGP20712A, black. (Scale bar: 10 μm.)

It was surprising to find that a major fraction of the phosphorylation of S1928 could be attributed to activation of the β2-adrenergic receptor pathway, because this pathway makes a small contribution to regulation of whole-cell calcium currents in rodent cardiac myocytes (refs. 26 and 28–30, but see refs. 31 and 32). Whole-cell calcium currents are usually measured with a calcium chelator like 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) in the recording pipette. To test the effect of BAPTA on phosphorylation of S1928, we bathed dissociated ventricular myocytes in extracellular solution containing the membrane-permeable derivative BAPTA–acetoxymethyl ester (AM) (5 μM), a concentration that prevents calcium transients in cardiac myocytes (33). Contractile responses were inhibited by this concentration of BAPTA-AM, indicating effective buffering of intracellular calcium (data not shown). Stimulation of the dissociated myocytes with Iso in the presence of BAPTA-AM caused a substantial increase in phosphorylation of S1928, as in the absence of BAPTA-AM (Fig. 4 I and O). However, nearly all of the Iso-induced increase in phosphorylation of S1928 was blocked by the β1-selective antagonist CGP 20712A (Fig. 4 J and O), whereas the β2-selective antagonist ICI 118551 had no detectable effect (Fig. 4 K and O). These results indicate that the stimulation of phosphorylation of S1928 by the β2-adrenergic pathway requires elevation of intracellular calcium above basal levels, whereas stimulation by the β1-adrenergic pathway does not.

To quantitatively correlate phosphorylation of S1928 with regulation of the calcium current, we measured Iso stimulation of calcium currents without and with selective β-adrenergic antagonists in the presence of intracellular BAPTA. As expected (18, 34), Iso treatment increased the calcium current 3.34- ± 0.2-fold (Fig. 5A). In agreement with our immunocytochemistry results with BAPTA-AM, we found that the β1-adrenergic antagonist CGP 20712A nearly completely inhibited the Iso stimulation of calcium current (98.7 ± 0.1%, Fig. 5B), whereas the β2-adrenergic antagonist ICI 118551 had little effect (3.6 ± 0.3%, Fig. 5C). Application of both antagonists completely prevented Iso stimulation (Fig. 5D). Therefore, our results show a close correlation between β1-adrenergic-stimulated phosphorylation of S1928 and stimulation of the calcium current when intracellular calcium is buffered.

Fig. 5.

Fig. 5.

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Effect of CGP 20712A and ICI 118551 on modulation of the Iso-stimulated calcium current in rat ventricular myocytes. (A–D Left) Representative calcium currents elicited by 200-ms test pulses from −40 to 10 mV before and after 5 min exposure to 100 nM Iso. (A–D Right) Mean (±SEM) current–voltage relationships before (filled symbols) and after (open symbols) a 5-min exposure to Iso. (A) The effect of Iso on the calcium current in the absence of inhibitors of β-adrenergic receptors. (B–D) The effect of a 20-min pretreatment with CGP 20712A (B; 1 μM, n = 6), ICI 118551 (C; 1 μM, n = 6), and CGP 20712A plus ICI 118551 (D; 1 μM, n = 6) on the response to Iso compared with the response to Iso without inhibitors (dotted line).

Discussion

Phosphorylation of S1928 During β-Adrenergic Regulation of Cardiac Calcium Channels.

Activation of adenylyl cyclase and the resulting increase in intracellular cAMP cause a striking increase in phosphorylation of S1928 of Cav1.2a channels expressed in tsA-201 cells, as measured by anti-CH3P in immunoblots and immunocytochemistry, but no phosphorylation is observed for Cav1.2a channels in which S1928 has been mutated to alanine. These results confirm the specificity of anti-CH3P for binding to phosphorylated S1928. Using this well characterized antibody, we found that Iso increases phosphorylation of S1928 in rat ventricular myocytes. This increase is mimicked when adenylyl cyclase is stimulated with forskolin. These results establish that S1928 is phosphorylated in concert with regulation of Cav1.2 channels by the β1-adrenergic/PKA pathway in intact cardiac myocytes.

Stimulation of Phosphorylation of S1928 by the β1- and β2-Adrenergic Pathways.

Phosphorylation of S1928 was stimulated substantially by both the β1- and β2-adrenergic pathways in physiological extracellular solution, as assessed with specific β-adrenergic antagonists at saturating concentrations. Quantitation by line scanning revealed that β1-adrenergic signaling was required for 65% of the phosphorylation of S1928 and β2-adrenergic signaling was required for 35%. In contrast, buffering intracellular calcium with BAPTA-AM prevented β2-adrenergic stimulation of phosphorylation. These results suggest that β2-adrenergic receptor-stimulated intracellular calcium transients are required for phosphorylation of S1928 in intact ventricular myocytes. Therefore, this amino acid residue may be phosphorylated by calcium-regulated protein kinases such as protein kinase C and calcium/calmodulin-dependent protein kinase II in intact ventricular myocytes, as observed in vitro for protein kinase C (35). β2-adrenergic receptor signaling engages Gi-stimulated (36) and β-arrestin-stimulated pathways (37) that are not engaged by β1-adrenergic signaling, which is specific for cAMP and PKA (38). These pathways may lead to increased intracellular calcium and phosphorylation of S1928 by protein kinase C and/or calcium/calmodulin-dependent protein kinase II, which would act in concert with β1-adrenergic stimulated phosphorylation of S1928 in regulating Cav1.2 channels.

Regulation of Cardiac Calcium Currents by the β1- and β2-Adrenergic Pathways.

Previous results have shown that the increase in whole-cell calcium currents in acutely dissociated ventricular myocytes by Iso and other nonselective β-adrenergic agonists is predominantly caused by activation of the β1-adrenergic receptor in rat, mouse, and dog (refs. 28 and 39–41, but see refs. 31 and 32), and we found that >98% of the β-adrenergic response is caused by activation of the β1-adrenergic pathway in rat ventricular myocytes. In contrast to these results with whole-cell voltage clamp, significant regulation of Cav1.2 channels in cell-attached patches on intact rat ventricular myocytes can be observed when β2-adrenergic receptors are locally activated with β2-selective agents in the recording pipette (32, 42). This apparent specificity of receptor action is mediated in part by localized interactions of adenylyl cyclase and β2-adrenergic receptors with Cav1.2 channels (32, 42). These results have led to the concept that β2-adrenergic signaling specifically stimulates a small population of cardiac calcium channels located at the cell surface by local production of cAMP, whereas β1-adrenergic signaling stimulates calcium channels throughout the cell via a more general increase in cAMP (32, 42, 43). Our previous finding that anchored PKA interacting with AKAP15 is required for stimulation of cardiac calcium currents by Iso in rat ventricular myocytes (18) provides a molecular mechanism for focal regulation of Cav1.2 channels by local increases in cAMP. In neurons, localized regulation of Cav1.2 channels is thought to be mediated by a specific complex with PKA, the AKAP MAP2B, and β2-adrenergic receptors (42), and a similar complex of Cav1.2 channels and β2-adrenergic receptors is observed in caveolae in cardiac myocytes (41) and may contribute to local regulation of Cav1.2 channels there.

Regulation of CaV1.2 Channels by Protein Phosphorylation.

Previous experiments on reconstitution of regulation of Cav1.2 channels by activation of PKA in transfected cells have given relatively small and inconsistent responses, up to 1.5-fold in only a fraction of the cells (16, 44, 45), in contrast to the reliable 3- to 4-fold increases observed in ventricular myocytes. In one case, a 1.35-fold increase in channel activity was reduced by mutation of S1928 to alanine (45). In another case, mutation of S1928 to alanine in Cav1.2 channels expressed in Xenopus oocytes prevented reduction of basal calcium currents by PKA inhibitors, suggesting that basal phosphorylation of S1928 increases calcium channel activity (46). However, the small increase in Cav1.2 channel activity observed in these two studies does not allow the role of phosphorylation of S1928 in the much larger increases in Cav1.2 channel activity in ventricular myocytes to be definitively assessed.

An elegant recent study (47) has tested the role of phosphorylation of S1928 in regulation of Cav1.2 channels expressed from a viral vector in cardiac myocytes. Peak calcium current conducted by the transfected Cav1.2a channels was increased 1.9-fold (47), compared with 3- to 4-fold increases recorded for the native channels in ventricular myocytes. Deletion of the distal C-terminal domain prevented PKA regulation (47), consistent with our previous finding that anchoring of PKA and AKAP15 to the distal C terminus via a modified leucine-zipper interaction was required for β-adrenergic regulation (18). In contrast, transfected Cav1.2 channels having S1928 mutated to alanine could still be regulated by β-adrenergic stimulation (47). The level of stimulated calcium current was 70–80% of that observed with wild-type Cav1.2 channels, but this reduction of 20–30% in β-adrenergic regulation of the S1928A mutant did not reach statistical significance (47). Therefore, these results indicate that phosphorylation of S1928 is not required for significant β-adrenergic regulation, but they leave room for up to 20–30% of the total response to be derived from phosphorylation of this amino acid residue.

Implications for the Role of Phosphorylation of S1928 in β-Adrenergic Regulation of CaV1.2 Channels.

The results of Ganesan et al. (47) on regulation of Cav1.2a channels with the S1928A mutation in virally infected cardiac myocytes show persuasively that phosphorylation of S1928 is not necessary for substantial stimulation by the β-adrenergic receptor pathway, our results indicate that phosphorylation of S1928 occurs during β-adrenergic regulation. Our finding that this residue is phosphorylated during activation of the β-adrenergic pathway, together with the suggestion of 20–30% lower stimulation of Cav1.2/S1928A channels in virally transfected ventricular myocytes (47), suggest the possibility that phosphorylation of this residue may enhance stimulation of calcium channel activity caused by phosphorylation of other site(s) on the Cav1.2 channel and may have other effects on channel expression, localization, degradation, or function.

Methods

Antibodies and cDNAs.

Rabbit polyclonal anti-CNC1 and anti-CH2 antibodies were generated against peptides corresponding to residues 821–838 in the intracellular loop of rbCII between domains II and III (CNC1) and residues 2051–2066 (CH2) in the distal C terminus of Cav1.2a, respectively, as described (9). Anti-CH3P, a phosphospecific antibody, was generated against a phosphopeptide (residues 1923–1932) that encompasses the PKA consensus site at S1928 as described (9). α11.2a/S1928A was generated by using PCR overlap extension, cloned into pcDNA3, and confirmed by DNA sequencing.

Expression of Wild-Type and S1928A CaV1.2 Channels.

TsA-201 cells were cultured in DMEM/Ham's F-12 supplemented with 10% FBS, 100 units/ml penicillin, and streptomycin and plated on 15-mm dishes. Cells were grown to ≈70% confluence and transfected with an equimolar ratio of cDNAs encoding wild-type α11.2a (48) or α11.2a/S1928A, β1b (49), and α2δ1 (50). To study the effect of PKA activation on phosphorylation of S1928 in cell culture, transfected tsA-201 cells were treated for 10 min with control culture medium or medium containing 100 nM okadaic acid plus 10 μM forskolin. Cells were solubilized in ice-cold bRIA (50 mM Tris·HCl, pH 7.4/150 mM NaCl/1 mg/ml BSA/5 mM NaF/1 mM EGTA/5 mM EDTA/1% Triton X-100, plus protease inhibitors) and rotated at 4°C for 30 min. Unsolubilized material was removed by centrifugation, and the solubilized extracts were separated by SDS/PAGE, transferred to nitrocellulose, and analyzed by immunoblotting. Total Cav1.2 channel protein was detected by using anti-CNC1 antibody. For detection of phosphorylated S1928, immunoblots were probed with anti-CH3P. Primary antibodies were detected by using horseradish peroxidase-linked protein A and ECL (Amersham Biosciences, Piscataway, NJ).

Cell Isolation.

Male Wistar rats (201–225 g) were killed by halothane overdose. Ventricular myocytes were isolated as described (51) and maintained at 37°C until use.

Electrophysiology.

Whole-cell calcium currents (ICa) were recorded at room temperature from rod-shaped, striated, calcium-tolerant myocytes within 1–8 h of isolation. The extracellular bath solution contained 140 mM tetraethylammonium·Cl, 2 mM MgCl2, 1.8 mM CaCl2, 10 mM Hepes, 10 mM glucose, and 20 μM tetrodotoxin at pH 7.4. Patch pipettes (1–1.5 MΩ) were filled with an intracellular solution (pH 7.3) containing 135 mM CsCl, 1 mM MgCl2, 5 mM MgATP, 10 mM Hepes, and 10 mM BAPTA. Series resistance ranged between 3 and 6 MΩ (60–80% compensated), and recordings were discarded if the series resistance was >6 MΩ before compensation. 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. The effect of Iso (100 nM) on ICa was examined 10 min after establishing the whole-cell configuration. For β-adrenergic receptor inhibition experiments, myocytes were incubated with 1 μM ICI 118551 or 1 μM CGP 20712A for 20 min at 37°C before recording. Currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) and sampled at 5 kHz after anti-alias filtering at 2 kHz. Data acquisition and command potentials were controlled by pClamp software v8.0 (Molecular Devices, Sunnyvale, CA), and data were stored for later off-line analysis. All data are expressed as the mean ± SE. Statistical significance was tested by using Student's t test. Values of P < 0.01 were considered significant.

Immunocytochemistry.

Ventricular myocytes were plated on laminin-coated glass coverslips and incubated (5% CO2, 37°C) for 2 h in isolation medium (51). Myocytes were rinsed and incubated for 10 min at room temperature in the absence or presence of 100 nM Iso, 10 μM forskolin, or 5 μM BAPTA-AM, a concentration that prevents calcium transients in cardiac myocytes (33). For β-adrenergic receptor inhibition experiments, myocytes were incubated with 1 μM ICI 118551 or 1 μM CGP 20712A for 20 min at 37°C before Iso treatment. Transfected tsA-201 cells were plated on laminin-coated glass coverslips and incubated (5% CO2, 37°C) for 24 h. For PKA stimulation experiments, cells were rinsed and then incubated in the absence or presence of 10 μM forskolin plus 100 nM okadaic acid for 10 min at room temperature. After rinsing, cells were fixed with 4% paraformaldehyde for 45 min, rinsed in 0.1 M phosphate, 0.1 M Tris buffer (TB), and 0.1 M Tris-buffered saline (TBS), then blocked sequentially with 2% avidin and 2% biotin-TBS. Myocytes were incubated with primary antibodies (diluted in TBS containing 0.25% Triton X-100 and 10% normal goat serum) overnight at 4°C. After extensive rinsing, cells were incubated with biotinylated antibody against rabbit IgG for 2 h at room temperature, rinsed, and then incubated with avidin D fluorescein (Vector Laboratories, Burlingame, CA) for 2 h at room temperature. After final washes, coverslips were mounted on slides by using Vectashield (Vector Laboratories). Cells were imaged by using a Bio-Rad (Hercules, CA) MRC 600 confocal microscope in the W. M. Keck Imaging Facility at the University of Washington.

Line Scan Analysis of Immunofluorescence Staining.

With the Igor computer program (Wavemetrics, Lake Oswego, OR), a 100-pixel-wide band was drawn across each myocyte, and the mean pixel intensity within the band was measured and plotted versus the distance across the myocyte. Means of these line-scan plots from multiple myocytes in a particular condition were generated by normalizing the width of the myocytes to the mean width of the myocytes in the sample and averaging. For each antibody used in a given experiment, the gain and offset were kept constant. The immunofluorescence of unstimulated cells was collected by using a 10% transmittance neutral density filter. Iso- and forskolin-stimulated cell immunofluorescence (in the absence of blockers) was typically collected by using a 2% transmittance neutral density filter to maintain signals in the most linear range and prevent saturation. The relative pixel intensity of staining was then multiplied by a correction factor of 5.

Abbreviations

AM

acetoxymethyl ester

BAPTA

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

Iso

isoproterenol

Footnotes

The authors declare no conflict of interest.

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