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Functional Regulation of L-type Calcium Channels via Protein Kinase A-mediated Phosphorylation of the β2 Subunit*

Activation of protein kinase A (PKA) through the β-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the β2 subunit are of functional significance, we coexpressed wild-type (WT) or mutant β2 subunits in tsA-201 cells together with an α1C subunit, α1CΔ1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated α1C subunit was similar to the truncated α1C subunit isolated from cardiac tissue not only in size (∼190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT β2 subunit, voltage-activated Ba2+ currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the β2 subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca2+currents may be at least partially caused by phosphorylation of the β2 subunit at Ser-478 and Ser-479.

It has been known for more than a decade that the cardiac L-type calcium channel is an important effector for positive modulation of cardiac contractility through signaling cascades initiated by activation of the ␤-adrenergic receptors (1). It is well accepted that activation of protein kinase A (PKA) 1 through the ␤AR pathway is crucial for the positive regulation of cardiac L-type Ca 2ϩ currents (1). Cardiac L-type Ca 2ϩ chan-nels are composed of ␣ 1C, ␤ 2 , and ␣ 2 ␦ subunits (2,3), and both the ␣ 1C and ␤ 2 subunits have been demonstrated to be direct targets of PKA-mediated phosphorylation (4 -7). However, it has been difficult to elucidate how the phosphorylation of each of these subunits might contribute to functional regulation of the channels in intact cells and to assign specific roles of the multiple sites of phosphorylation to specific functional changes in channel properties. Studies in intact cardiac myocytes are extremely difficult due to the low abundance of channel proteins, thus studies in heterologous expression systems have the potential to define the roles of subunit phosphorylation in the regulation of the channels. However, a problem with this approach is that it has been difficult to reconstitute in heterologous expression systems the robust regulation of L-type channels that is observed in cardiac cells (8).
Cyclic AMP-dependent phosphorylation and functional regulation of the channels was facilitated in human embryonic kidney cells when the channels were coexpressed with the protein kinase A-anchoring proteins AKAP79 and AKAP15/18 (6,9). While both the ␣ 1C and ␤ 2 subunits were phosphorylated when coexpressed with AKAP79, only phosphorylation of serine (Ser) 1928 in the pore-forming ␣ 1C subunit appeared to be functionally linked to channel regulation (6). However, compared with the robust PKA-mediated stimulation of native L-type currents (3-6-fold in many species), the effects of PKA in the heterologous expression systems in the presence of either AKAP were rather small (50% increase in peak currents) (6,9). In addition, Ser-1928 is present in a portion of the C terminus of the ␣ 1C subunit that appears to be subject to proteolytic processing in native systems (10,11), suggesting the possibility that this site may not be available to mediate PKA-mediated regulation in cardiac myocytes. This suggests that other events, such as phosphorylation of the ␤ subunit, may play a functional role in channel regulation. This goal of this study was to test whether or not PKA mediated phosphorylation of the ␤ 2 subunit has functional consequences. The ␤ 2 subunit has been shown to undergo cAMP-dependent phosphorylation at multiple sites in vitro and in cardiac myocytes and intact hearts (12)(13)(14). While the rat ␤ 2a subunit has two consensus sequences at Thr-164 and Ser-591 that might serve as PKA sites, these sites are not phosphorylated by PKA (14). Rather, the actual sites of PKA-mediated phosphorylation on the ␤ 2a subunit are Ser-459, Ser-478, and Ser-479 (14). An additional goal of this study was to determine which, if any, of these sites might mediate functional changes in channel activity. In order to prevent contributions from the previously identified PKA phosphorylation site on the ␣ 1C subunit and to mimic conditions that may exist in native systems, we utilized a truncation mutant of the ␣ 1C subunit, ␣ 1C ⌬1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928.

MATERIALS AND METHODS
Cell Culture-TsA-201 cells (large T antigen-transformed human embryonic kidney cells) were maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units of penicillin and streptomycin (Life Technologies, Inc.) in an humidified atmosphere containing 5% CO 2 . TsA cells were transiently cotransfected with the truncated ␣ 1C ⌬1905 subunit (rabbit) and either the wild-type (WT) rat ␤ 2a subunit or the S459A or S478A/S479A ␤ 2a mutants. Each construct was in the pCR3 vector and 3 g of each was used per 10-cm plate, along with the CD8 reporter vector, H3-CD8, (0.5 g/10-cm plate) (15), using the Effectene transfection kit following the manufacturer's recommended protocol (Qiagen). Cells were replated 24 -48 h following transfection on 3-cm plates that were previously coated with collagen (Sigma). Transfected cells were visualized by using the CD8 receptor as a reporter gene and marking transfected cells with anti-CD8 antibody coated Dynabeads (Dynal) (15).
In Vitro Phosphorylation of Calcium Channel Subunits by PKA-The wild-type ␣ 1C or the truncation mutant ␣ 1C ⌬1905 subunits were coexpressed with the WT ␤ 2a subunit in tsA-201 cells. Whole cell lysates were prepared from the transfected cells using lysis buffer (50 mM Tris, pH 7.4, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, and protease inhibitors (11)). The channel subunits were immunoprecipitated from the cell lysates using an ␣ 1C subunit-specific antibody, Card I, coupled to Ultra-Link protein G (Pierce). Native ␣ 1C subunits from rabbit heart were assayed in the phosphorylation experiments as well. To isolate the native ␣ 1C subunits, crude membranes from frozen rabbit heart were prepared. For immunoprecipitation, membranes were solubilized in lysis buffer and incubated with the Card I antibody coupled to protein G (11). The immunoprecipitated channel subunits from transfected tsA cells and rabbit heart were phosphorylated with the purified catalytic subunit of PKA following the procedures described previously (4). The phosphorylated channel proteins were separated by SDS-polyacrylamide gel electrophoresis followed by phosphorimaging and immunoblot analysis. Detection of the ␣ 1C subunits on the immunoblot was with biotinylated Card I (11).
Solutions-For the measurement of Ba 2ϩ currents through L-type calcium channels, the external solution consisted of 10 mM BaCl 2 , 105 mM NaCl, 25 mM CsCl, and 10 mM Hepes, pH 7.4. The pipette solution was composed of 100 mM cesium aspartate, 40 mM CsCl, 1 mM MgCl 2 , 2 mM Mg-ATP, 0.5 mM GTP, 5 mM EGTA, and 5 mM Hepes, pH 7.4. When present, PKA was included in the pipette at a final concentration of 20 nM.
Current Measurement-Ba 2ϩ currents through L-type calcium channels were measured in the whole cell configuration of the patch clamp technique using fire-polished borosilicate glass pipettes (GF-150 -10, Warner Instrument Corp.) generated with a horizontal puller (P-95 Fleming & Poulsen) with a final resistance between 2 and 4 megaohms. Membrane currents were recorded as described previously (16) using a patch clamp amplifier (Axopatch 200, Axon Instruments). Signals were analog-filtered using a low pass Bessel filter (1-3 kHz corner frequency). Data were digitally stored using an IBM-compatible PC equipped with a hardware/software package (ISO2 by MFK) for voltage control, data acquisition, and data evaluation.
After establishing the whole cell configuration, cells were clamped at Ϫ90 mV and voltage pulses (test pulses) (100-ms duration) to ϩ10 mV were applied every 10 s in order to activate L-type calcium channels. After reaching a steady state of the current amplitude (define as current amplitude that did not change in three subsequent test pulses), which took 3-15 min, the current-voltage relationship (I-V curve) was measured by varying the potential of the test pulses from Ϫ80 to ϩ50 mV. Capacitative currents due to recharging the cell membrane were compensated. Currents were normalized to capacitance.
The catalytic subunit of protein kinase A was purified to homogeneity from bovine heart (17). In all experiments in which the effect of PKA was studied, control experiments (without PKA) were performed in parallel in order to minimize variations in current amplitudes due to differences from transfection to transfection. The summarized data were pooled from at least three different transfections, unless otherwise noted and are expressed as mean Ϯ S.E.

RESULTS
Can PKA regulate channels lacking a phosphorylatable ␣ 1C subunit? The first question we addressed was whether or not PKA can regulate cardiac L-type calcium channels comprised of a wild-type ␤ 2 subunit and a truncated ␣ 1C subunit that lacks Ser-1928, the site that was previously shown to be phosphorylated both in vitro (4,5) and in intact cells (6). The truncated ␣ 1C subunit used in these studies contained a deletion that resulted in the loss of its C terminus downstream of residue 1905 (␣ 1C ⌬1905). This mutant ␣ 1C ⌬1905 subunit had a similar molecular mass when analyzed by SDS-polyacrylamide gel electrophoresis as the truncated ␣ 1C subunit isolated from cardiac tissue (Fig. 1). Importantly, ␣ 1C ⌬1905 was not a substrate for PKA (Fig. 1), confirming that Ser-1928 is the sole site phosphorylated by PKA in the ␣ 1C subunit (4, 6). Representative voltagedependent Ba 2ϩ currents through channels consisting of ␣ 1C ⌬1905 and WT ␤ 2 subunits were elicited in response to depolarization to different test potentials. The current voltage relationship exhibited the typical properties of Ba 2ϩ currents through L-type Ca channels (Fig. 2).
In order to test the effects of PKA on these currents, the purified catalytic subunit of PKA was added to the patch pipette at a final concentration of 20 nM. This resulted in an approximately two-fold increase (from Ϫ47.5 Ϯ 11.4 pA/pF to Ϫ116.2 Ϯ 26.2 pA/pF at 0 mV test potential, n ϭ 10 -11) in current amplitude of the Ba 2ϩ current generated by the channels formed by the ␣ 1C ⌬1905 and the WT ␤ 2a subunits (Fig. 2,  A and B). These results demonstrated that PKA could indeed cause increases in currents generated from channels lacking a phosphorylatable ␣ 1C subunit. In addition, the 2-fold increase in peak current amplitude resembled that seen in native cardiac myocytes. On the other hand, no apparent hyperpolarizing shift in the current-voltage (I-V) curve of the Ba 2ϩ current was observed. This latter effect is routinely observed in native cardiac myocytes.
In order to test whether or not the PKA-mediated increase in Ba 2ϩ currents was due to phosphorylation of the ␤ 2a subunit, we expressed the ␣ 1C ⌬1905 subunit with mutant ␤ 2a subunits that lacked the identified PKA phosphorylation sites (14). These mutants contained point mutations of serines to alanines either at position 459 or 478/479 (14). Voltage-dependent Ba 2ϩ currents in cells expressing ␣ 1C ⌬1905 and ␤ 2a S459A were indistinguishable from currents obtained in cells expressing the WT ␤ 2 subunit (Fig. 3), indicating that this mutation did not alter the basic functional properties of the regulatory ␤ 2a subunit. The addition of the catalytic subunit of PKA to the patch pipette caused a significant increase in Ba 2ϩ currents compared with the controls (Ϫ138.1 Ϯ 37.7 pA/pF versus Ϫ28.2 Ϯ 7.0 pA/pF at 0 mV, n ϭ 6 -7) in cells expressing ␣ 1C ⌬1905 and the mutant ␤ 2a S459A (Fig. 3, A and B). This effect was comparable with that observed with the WT ␤ 2a subunit (compare Fig. 2 with Fig. 3). In addition and in contrast to the results obtained with the WT ␤ 2a subunit, a significant (p Ͻ 0.05) shift of the voltage that caused half-maximal activation of calcium channels from Ϫ7.8 Ϯ 2.4 mV to Ϫ14.4 Ϯ 0.6 mV (n ϭ 6 -7) was observed in response to PKA by analysis of steady state activation curves (Boltzmann fit).
The channels formed by ␣ 1C ⌬1905 and ␤ 2a S478A/S479A also produced currents that were similar in current density and voltage dependence to those obtained with the WT ␤ subunit in the absence of PKA (Fig. 4 compared with Fig. 2), indicating that these point mutations did not lead to gross misfolding of FIG. 1. In vitro phosphorylation of the L-type calcium channel ␣ 1c subunits. The wild-type ␣ 1C and the truncation mutant ␣ 1C ⌬1905 subunits were immunoprecipitated from transfected tsA-201 cells, while the native ␣ 1C subunits were isolated from rabbit heart as described under "Materials and Methods." The immunoprecipitated channel subunits were subjected to in vitro phosphorylation by purified PKA. A representative phosphorimage and the corresponding immunoblot are shown. Note only the wild-type ␣ 1C subunit was phosphorylated by PKA.
the ␤ subunit protein. However, in marked contrast to what was observed with the WT and S459A mutant ␤ subunits, the addition of PKA to the pipette did not augment currents obtained with ␣ 1C ⌬1905 and the S478A/S479A ␤ 2a subunit (Ϫ59.1 Ϯ 12.1 pA/pF versus Ϫ63.5 Ϯ 12.4 pA/pF at 0 mV, n ϭ 9). We have previously demonstrated that Ser-478 and Ser-479 are key residues for phosphorylation by PKA and that mutation of these two serines to alanines causes a 75% reduction in the PKA-mediated phosphorylation of the ␤ 2 subunit. Taken together with the fact that the ␣ 1C ⌬1905 subunit was not a substrate for PKA, the functional effects of the PKA-mediated regulation seen in the studies reported here are likely to occur through phosphorylation of the ␤ 2a subunit. These results demonstrated the importance of phosphorylation of the ␤ 2a subunit to the regulation of the cardiac calcium channel and identified the functionally important residues in the ␤ 2 subunit that are important for channel regulation. DISCUSSION The regulation of the cardiac L-type calcium channel by activation of PKA has been extremely well characterized through electrophysiological studies (1); however the underlying phosphorylation reactions have not been resolved completely. In particular, the substrates for PKA that are responsible for the stimulation of the calcium current in intact cardiac myocytes are unknown. The results shown here, together with those in recent companion studies (6,7,14), give new insights into this process and demonstrate that the regulation of the channel may occur through more than one process. While early studies encountered difficulties in obtaining PKA-mediated stimulation of the cardiac L-type channel in various heterologous expression systems (8), we now have learned of two scenarios that will allow for expression of the PKA effects. In studies with full-length ␣ 1C and ␤ 2a subunits, cAMP-dependent effects can be observed only if the channels are co-expressed with an AKAP (6). In this scenario, the cAMP-dependent effects were attributed to phosphorylation of Ser-1928 in the C terminus of the ␣ 1C subunit, as mutation of this site alone led to a loss of the PKA effect (6). In addition, the PKA-mediated phos- phorylation of Ser-1928 in the ␣ 1C subunit was AKAP-dependent, while phosphorylation of the ␤ 2a subunit was not (6). Thus, even though the ␤ 2a was phosphorylated at multiple sites when it was coexpressed with the full-length ␣ 1C subunit in the presence or absence of an AKAP, there did not appear to be a functional consequence of the phosphorylation of the ␤ 2a subunit (6). Interestingly, in this scenario, the increases in peak current were small, but a significant hyperpolarizing shift in the current-voltage relationship was observed (6). In the second scenario reported here, in studies with a C-terminally truncated, nonphosphorylatable ␣ 1C subunit, we have demonstrated the functional importance of the phosphorylation of two adjacent sites at Ser-478 and Ser-479 in the ␤ 2 subunit for regulation of the channel in response to PKA. In this second scenario, the increase in peak current was more substantial than observed in the first scenario, but a small hyperpolarizing shift in the current-voltage relationship was only observed in the context of the ␤ 2a S459A mutant. These effects did not require the expression of an AKAP, in agreement with the previous observation that the ␤ 2a subunit could undergo PKAdependent phosphorylation whether or not it was co-expressed with an AKAP. These results contribute new aspects to mechanisms of regulation of the cardiac L-type channel, in particular that the ␤ subunit may be directly involved in the regulatory process. Further studies are necessary to define whether one or both, or even other, events contribute to the PKAmediated regulation of the channels in intact myocytes. Since neither scenario alone exhibits both the large increases in peak current amplitude and the hyperpolarizing shift in the currentvoltage relationship that are observed in native cardiac myocytes, it is possible that both scenarios contribute to current regulation in the heart.
Key to understanding exactly what modes of regulation exist in cardiac myocytes is to elucidate the status and role of the ␣ 1C C terminus. It is possible that the truncation of the ␣ 1C subunit is necessary to allow for expression of the functional consequences of PKA-mediated phosphorylation of the ␤ 2 subunit and that regulation proceeds through a different mechanism when the full-length ␣ 1C subunit is the major form present. Yet another mechanism of regulation of the channel may be possible if the ␣ 1C subunit is cleaved in intact cells and the Cterminal fragment remains functionally associated with the "body" of the channel. This latter possibility is suggested by the observations that ϳ85-90% of the ␣ 1C subunit appears to be truncated at the C terminus when biochemically isolated from native tissues, yet immunofluorescent studies suggest that the C terminus is present in cardiac myocytes in stoichiometric amounts and co-localized with the ␣ 1C and ␤ 2 subunits (11). Potentially the truncation of the C terminus may allow new conformations of the channel to exist and alter the functional consequences of the phosphorylation of both the ␣ 1C and ␤ 2 subunits. Future studies will address these potentially complex mechanisms of channel regulation and further probe the types of regulation that occur in native systems.