Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac CaV1.2 channels during beta1-adrenergic regulation.
During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by Ca(V)1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of beta-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the alpha(1) subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to beta-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 beta1- and beta2-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 beta1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to beta1-adrenergic receptors. Thus, beta-adrenergic stimulation results in phosphorylation of S1928 on the Ca(V)1.2 alpha1 subunit in intact ventricular myocytes via both beta1- and beta2-adrenergic receptor pathways, but the beta2-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 beta1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.
Hulme
Joanne T
JT
Department of Pharmacology, University of Washington, Mailstop 357280, Seattle, WA 98195-7280, USA.
Westenbroek
Ruth E
RE
Scheuer
Todd
T
Catterall
William A
WA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018342:Receptors, Adrenergic, beta-1 / Q000378:metabolism*
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D018342:Receptors, Adrenergic, beta-1; D010768:Phosphoserine; D005576:Colforsin; D002118:Calcium