Functional Regulation of L-type Calcium Channels via Protein Kinase A-mediated Phosphorylation of the β2 Subunit
1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês
10.1074/jbc.274.48.33851
ISSN1083-351X
AutoresMoritz Bünemann, Brian L. Gerhardstein, Tianyan Gao, Marie Thérèse Hosey,
Tópico(s)Receptor Mechanisms and Signaling
ResumoActivation 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. 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. protein kinase A wild-type farad(s) A-kinase-anchoring protein 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 β-adrenergic receptors (1McDonald T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (923) Google Scholar). It is well accepted that activation of protein kinase A (PKA)1 through the βAR pathway is crucial for the positive regulation of cardiacl-type Ca2+ currents (1McDonald T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (923) Google Scholar). Cardiac L-type Ca2+ channels are composed of α1C,β2, and α2δ subunits (2De Waard M. Gurnett C.A. Campbell K.P. Narahashi T. Ion Channels. 4. Plenum Press, New York1996: 41-87Google Scholar, 3Hosey M.M. Chien A.J. Puri T.S. Trends Cardiovasc. Med. 1996; 6: 265-273Crossref PubMed Scopus (47) Google Scholar), and both the α1C and β2 subunits have been demonstrated to be direct targets of PKA-mediated phosphorylation (4De Jongh K.S. Murphy B.J. Colvin A.A. Hell J.W. Takahashi M. Catterall W.A. Biochemistry. 1996; 35: 10392-103402Crossref PubMed Scopus (239) Google Scholar, 5Mitterdorfer J. Froschmayr M. Grabner M. Moebius F.F. Glossmann H. Striessnig J. Biochemistry. 1996; 35: 9400-9406Crossref PubMed Scopus (82) Google Scholar, 6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 7Puri T.S. Gerhardstein B.L. Zhao X.L. Ladner M.B. Hosey M.M. Biochemistry. 1997; 36: 9605-9615Crossref PubMed Scopus (108) Google Scholar). 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 (8Zong X. Schreieck J. Mehrke G. Welling A. Schuster A. Bosse E. Flockerzi V. Hofmann F. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 340-347Crossref PubMed Scopus (80) Google Scholar).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 (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 9Fraser I.D. Tavalin S.J. Lester L.B. Langeberg L.K. Westphal A.M. Dean R.A. Marrion N.V. Scott J.D. EMBO J. 1998; 17: 2261-2272Crossref PubMed Scopus (250) Google Scholar). While both the α1C and β2 subunits were phosphorylated when coexpressed with AKAP79, only phosphorylation of serine (Ser) 1928 in the pore-forming α1C subunit appeared to be functionally linked to channel regulation (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). 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) (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 9Fraser I.D. Tavalin S.J. Lester L.B. Langeberg L.K. Westphal A.M. Dean R.A. Marrion N.V. Scott J.D. EMBO J. 1998; 17: 2261-2272Crossref PubMed Scopus (250) Google Scholar). In addition, Ser-1928 is present in a portion of the C terminus of the α1C subunit that appears to be subject to proteolytic processing in native systems (10Chang F.C. Hosey M.M. J. Biol. Chem. 1988; 263: 18929-18937Abstract Full Text PDF PubMed Google Scholar, 11Gao T. Puri T.S. Gerhardstein B.L. Chien A.J. Green R.D. Hosey M.M. J. Biol. Chem. 1997; 272: 19401-19407Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), 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 β 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 β2 subunit has functional consequences. The β2 subunit has been shown to undergo cAMP-dependent phosphorylation at multiple sites in vitro and in cardiac myocytes and intact hearts (12Haase H. Karczewski P. Beckert R. Krause E.G. FEBS Lett. 1993; 335: 217-222Crossref PubMed Scopus (93) Google Scholar, 13Haase H. Bartel S. Karczewski P. Morano I. Krause E.G. Mol. Cell. Biochem. 1996; 163–164: 99-106Crossref PubMed Scopus (56) Google Scholar, 14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). While the rat β2a subunit has two consensus sequences at Thr-164 and Ser-591 that might serve as PKA sites, these sites are not phosphorylated by PKA (14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). Rather, the actual sites of PKA-mediated phosphorylation on the β2a subunit are Ser-459, Ser-478, and Ser-479 (14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). 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 α1C subunit and to mimic conditions that may exist in native systems, we utilized a truncation mutant of the α1C subunit, α1CΔ1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928.RESULTSCan PKA regulate channels lacking a phosphorylatable α1C subunit? The first question we addressed was whether or not PKA can regulate cardiac L-type calcium channels comprised of a wild-type β2 subunit and a truncated α1Csubunit that lacks Ser-1928, the site that was previously shown to be phosphorylated both in vitro (4De Jongh K.S. Murphy B.J. Colvin A.A. Hell J.W. Takahashi M. Catterall W.A. Biochemistry. 1996; 35: 10392-103402Crossref PubMed Scopus (239) Google Scholar, 5Mitterdorfer J. Froschmayr M. Grabner M. Moebius F.F. Glossmann H. Striessnig J. Biochemistry. 1996; 35: 9400-9406Crossref PubMed Scopus (82) Google Scholar) and in intact cells (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). The truncated α1C subunit used in these studies contained a deletion that resulted in the loss of its C terminus downstream of residue 1905 (α1CΔ1905). This mutant α1CΔ1905 subunit had a similar molecular mass when analyzed by SDS-polyacrylamide gel electrophoresis as the truncated α1C subunit isolated from cardiac tissue (Fig.1). Importantly, α1CΔ1905 was not a substrate for PKA (Fig. 1), confirming that Ser-1928 is the sole site phosphorylated by PKA in the α1C subunit (4De Jongh K.S. Murphy B.J. Colvin A.A. Hell J.W. Takahashi M. Catterall W.A. Biochemistry. 1996; 35: 10392-103402Crossref PubMed Scopus (239) Google Scholar,6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Representative voltage-dependent Ba2+currents through channels consisting of α1CΔ1905 and WT β2 subunits were elicited in response to depolarization to different test potentials. The current voltage relationship exhibited the typical properties of Ba2+ currents through L-type Ca channels (Fig. 2).Figure 2PKA-mediated regulation of L-type calcium currents. A, the voltage dependence of Ba2+currents through L-type calcium channels in cells cotransfected with the truncated α1CΔ1905 and the WT β2awere measured. Representative current traces obtained from a cell in response to test potentials of −80, −40, −20, −10, 0, 10, 20, 30, 50 mV, with or without PKA (20 nm) in the (intracellular) pipette solution, are shown. B, current-voltage relationships (I-V curves) for the peak IBaobtained from 10–11 cells from four independent transfections in the presence (filled circles) or absence (open circles) of PKA (20 nm) in the pipette solutions are shown.View Large Image Figure ViewerDownload (PPT)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 −47.5 ± 11.4 pA/pF to −116.2 ± 26.2 pA/pF at 0 mV test potential, n = 10–11) in current amplitude of the Ba2+ current generated by the channels formed by the α1CΔ1905 and the WT β2a subunits (Fig. 2, A and B). These results demonstrated that PKA could indeed cause increases in currents generated from channels lacking a phosphorylatable α1C 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 Ba2+ 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 Ba2+ currents was due to phosphorylation of the β2a subunit, we expressed the α1CΔ1905 subunit with mutant β2a subunits that lacked the identified PKA phosphorylation sites (14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). These mutants contained point mutations of serines to alanines either at position 459 or 478/479 (14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). Voltage-dependent Ba2+ currents in cells expressing α1CΔ1905 and β2aS459A were indistinguishable from currents obtained in cells expressing the WT β2 subunit (Fig. 3), indicating that this mutation did not alter the basic functional properties of the regulatory β2a subunit. The addition of the catalytic subunit of PKA to the patch pipette caused a significant increase in Ba2+ currents compared with the controls (−138.1 ± 37.7 pA/pF versus −28.2 ± 7.0 pA/pF at 0 mV, n = 6–7) in cells expressing α1CΔ1905 and the mutant β2a S459A (Fig.3, A and B). This effect was comparable with that observed with the WT β2a subunit (compare Fig. 2 with Fig. 3). In addition and in contrast to the results obtained with the WT β2a subunit, a significant (p < 0.05) shift of the voltage that caused half-maximal activation of calcium channels from −7.8 ± 2.4 mV to −14.4 ± 0.6 mV (n = 6–7) was observed in response to PKA by analysis of steady state activation curves (Boltzmann fit).Figure 3PKA-mediated stimulation of Ba2+currents through channels lacking phosphorylation site Ser-459 on the β2a subunit. A, representative current traces in response to different test potentials (see Fig. 1) obtained from cells expressing S459A β2asubunits with the α1CΔ1905 subunit with or without 20 nm PKA in the pipette (as indicated). B, summarized data for the voltage dependence of peak IBaobtained from similar experiments as described in A(n = 6–7).View Large Image Figure ViewerDownload (PPT)The channels formed by α1CΔ1905 and β2aS478A/S479A also produced currents that were similar in current density and voltage dependence to those obtained with the WT β subunit in the absence of PKA (Fig. 4 compared with Fig.2), indicating that these point mutations did not lead to gross misfolding of the β subunit protein. However, in marked contrast to what was observed with the WT and S459A mutant β subunits, the addition of PKA to the pipette did not augment currents obtained with α1CΔ1905 and the S478A/S479A β2a subunit (−59.1 ± 12.1 pA/pF versus −63.5 ± 12.4 pA/pF at 0 mV, n = 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 β2 subunit. Taken together with the fact that the α1CΔ1905 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 β2a subunit. These results demonstrated the importance of phosphorylation of the β2asubunit to the regulation of the cardiac calcium channel and identified the functionally important residues in the β2 subunit that are important for channel regulation.Figure 4Lack of PKA-mediated stimulation of L-type calcium channels in cells expressing S478A/S479A β2a subunits.Experiments were performed as described for Fig. 1 using the S478A/S479A β2a subunits instead of β2a WT subunits. A, representative barium currents obtained in response to different test potentials with or without 20 nm PKA in the internal solution (as indicated).B, summarized data for the I-V curves for voltage-activated I Ba measured in the absence or presence of PKA in the internal solution (n = 9).View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe regulation of the cardiac L-type calcium channel by activation of PKA has been extremely well characterized through electrophysiological studies (1McDonald T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (923) Google Scholar); 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 (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 7Puri T.S. Gerhardstein B.L. Zhao X.L. Ladner M.B. Hosey M.M. Biochemistry. 1997; 36: 9605-9615Crossref PubMed Scopus (108) Google Scholar, 14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar), 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 (8Zong X. Schreieck J. Mehrke G. Welling A. Schuster A. Bosse E. Flockerzi V. Hofmann F. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 340-347Crossref PubMed Scopus (80) Google Scholar), we now have learned of two scenarios that will allow for expression of the PKA effects. In studies with full-length α1C and β2a subunits, cAMP-dependent effects can be observed only if the channels are co-expressed with an AKAP (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). In this scenario, the cAMP-dependent effects were attributed to phosphorylation of Ser-1928 in the C terminus of the α1C subunit, as mutation of this site alone led to a loss of the PKA effect (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). In addition, the PKA-mediated phosphorylation of Ser-1928 in the α1C subunit was AKAP-dependent, while phosphorylation of the β2a subunit was not (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Thus, even though the β2a was phosphorylated at multiple sites when it was coexpressed with the full-length α1C subunit in the presence or absence of an AKAP, there did not appear to be a functional consequence of the phosphorylation of the β2asubunit (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Interestingly, in this scenario, the increases in peak current were small, but a significant hyperpolarizing shift in the current-voltage relationship was observed (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). In the second scenario reported here, in studies with a C-terminally truncated, nonphosphorylatable α1C subunit, we have demonstrated the functional importance of the phosphorylation of two adjacent sites at Ser-478 and Ser-479 in the β2 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 β2aS459A mutant. These effects did not require the expression of an AKAP, in agreement with the previous observation that the β2asubunit could undergo PKA-dependent 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 β 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 PKA-mediated 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 current-voltage 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 α1C C terminus. It is possible that the truncation of the α1Csubunit is necessary to allow for expression of the functional consequences of PKA-mediated phosphorylation of the β2subunit and that regulation proceeds through a different mechanism when the full-length α1C subunit is the major form present. Yet another mechanism of regulation of the channel may be possible if the α1C subunit is cleaved in intact cells and the C-terminal fragment remains functionally associated with the "body" of the channel. This latter possibility is suggested by the observations that ∼85–90% of the α1C 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 α1C and β2 subunits (11Gao T. Puri T.S. Gerhardstein B.L. Chien A.J. Green R.D. Hosey M.M. J. Biol. Chem. 1997; 272: 19401-19407Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). 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 α1C and β2 subunits. Future studies will address these potentially complex mechanisms of channel regulation and further probe the types of regulation that occur in native systems. 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 β-adrenergic receptors (1McDonald T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (923) Google Scholar). It is well accepted that activation of protein kinase A (PKA)1 through the βAR pathway is crucial for the positive regulation of cardiacl-type Ca2+ currents (1McDonald T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (923) Google Scholar). Cardiac L-type Ca2+ channels are composed of α1C,β2, and α2δ subunits (2De Waard M. Gurnett C.A. Campbell K.P. Narahashi T. Ion Channels. 4. Plenum Press, New York1996: 41-87Google Scholar, 3Hosey M.M. Chien A.J. Puri T.S. Trends Cardiovasc. Med. 1996; 6: 265-273Crossref PubMed Scopus (47) Google Scholar), and both the α1C and β2 subunits have been demonstrated to be direct targets of PKA-mediated phosphorylation (4De Jongh K.S. Murphy B.J. Colvin A.A. Hell J.W. Takahashi M. Catterall W.A. Biochemistry. 1996; 35: 10392-103402Crossref PubMed Scopus (239) Google Scholar, 5Mitterdorfer J. Froschmayr M. Grabner M. Moebius F.F. Glossmann H. Striessnig J. Biochemistry. 1996; 35: 9400-9406Crossref PubMed Scopus (82) Google Scholar, 6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 7Puri T.S. Gerhardstein B.L. Zhao X.L. Ladner M.B. Hosey M.M. Biochemistry. 1997; 36: 9605-9615Crossref PubMed Scopus (108) Google Scholar). 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 (8Zong X. Schreieck J. Mehrke G. Welling A. Schuster A. Bosse E. Flockerzi V. Hofmann F. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 340-347Crossref PubMed Scopus (80) Google Scholar). 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 (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 9Fraser I.D. Tavalin S.J. Lester L.B. Langeberg L.K. Westphal A.M. Dean R.A. Marrion N.V. Scott J.D. EMBO J. 1998; 17: 2261-2272Crossref PubMed Scopus (250) Google Scholar). While both the α1C and β2 subunits were phosphorylated when coexpressed with AKAP79, only phosphorylation of serine (Ser) 1928 in the pore-forming α1C subunit appeared to be functionally linked to channel regulation (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). 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) (6Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 9Fraser I.D. Tavalin S.J. Lester L.B. Langeberg L.K. Westphal A.M. Dean R.A. Marrion N.V. Scott J.D. EMBO J. 1998; 17: 2261-2272Crossref PubMed Scopus (250) Google Scholar). In addition, Ser-1928 is present in a portion of the C terminus of the α1C subunit that appears to be subject to proteolytic processing in native systems (10Chang F.C. Hosey M.M. J. Biol. Chem. 1988; 263: 18929-18937Abstract Full Text PDF PubMed Google Scholar, 11Gao T. Puri T.S. Gerhardstein B.L. Chien A.J. Green R.D. Hosey M.M. J. Biol. Chem. 1997; 272: 19401-19407Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), 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 β 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 β2 subunit has functional consequences. The β2 subunit has been shown to undergo cAMP-dependent phosphorylation at multiple sites in vitro and in cardiac myocytes and intact hearts (12Haase H. Karczewski P. Beckert R. Krause E.G. FEBS Lett. 1993; 335: 217-222Crossref PubMed Scopus (93) Google Scholar, 13Haase H. Bartel S. Karczewski P. Morano I. Krause E.G. Mol. Cell. Biochem. 1996; 163–164: 99-106Crossref PubMed Scopus (56) Google Scholar, 14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). While the rat β2a subunit has two consensus sequences at Thr-164 and Ser-591 that might serve as PKA sites, these sites are not phosphorylated by PKA (14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). Rather, the actual sites of PKA-mediated phosphorylation on the β2a subunit are Ser-459, Ser-478, and Ser-479 (14Gerhardstein B.L. Puri T.S. Chien A.J. Hosey M.M. Biochemistry. 1999; 38: 10361-10370Crossref PubMed Scopus (114) Google Scholar). 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 α1C subunit and to mimic conditions that may exist in native systems, we utilized a truncation mutant of the α1C subunit, α1CΔ1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. RESULTSCan PKA regulate channels lacking a phosphorylatable α1C subunit? The first question we addressed was whether or not PKA can regulate cardiac L-type calcium channels comprised of a wild-type β2 subunit and a truncated α1Csubunit that lacks Ser-1928, the site that was previously shown to be pho
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