Auxiliary beta subunits differentially determine pka utilization of distinct regulatory sites on Cav1.3 L type Ca2+ channels.
L-type calcium channels (Ca(v)1.1-Ca(v)1.4) link Ca(2+) influx to membrane depolarization and serve a critical role in regulating membrane excitability, muscle contraction, hormone secretion, and gene transcription. In many tissues, L-type calcium channel activity (Ca(v)1.1 and Ca(v)1.2) is enhanced by transmitters and hormones that activate the cAMP-dependent protein kinase (PKA), which is largely thought to be mediated via phosphorylation of the pore forming alpha subunit. However, the ability of PKA to regulate Ca(v)1.3 and the sites contributing to effective modulation of channel activity remains to be established. Using HEK 293 cells, we demonstrate that currents carried by the long C-terminal splice variant of Ca(v)1.3 (Ca(v)1.3L) are selectively enhanced compared to the short C-terminal splice variant (Ca(v)1.3S) when the catalytic subunit of PKA is introduced into the cell via the whole-cell recording electrode. However, the persistence of this regulation is dependent on the identity of the auxiliary beta subunit, such that PKA produces only a transient increase in the presence of beta(3) while a persistent increase is observed in the presence of the beta(2a) subunit. Site-directed mutagenesis of consensus PKA phosphorylation sites revealed that Ser1964 and Ser1743 in Ca(v)1.3L were the predominant sites controlling PKA modulation in the presence of the beta(3) and beta(2a) auxiliary subunits, respectively. Therefore, beta subunits determine the contribution of distinct sites within Ca(v)1.3 towards PKA-mediated enhancement of channel activity. These data suggest that auxiliary beta subunits govern the access of signaling enzymes to L-type calcium channels.
Liang
Yixin
Y
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Tavalin
Steven J
SJ
D000818:Animals; D001921:Brain / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006325:Heart Atria / Q000737:chemistry; D006352:Heart Ventricles / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000166:cytology; D009206:Myocardium / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D010957:Plasmids; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D020746:Calcium Channels, L-Type; D018076:DNA, Complementary; D020033:Protein Isoforms; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases