Abstract
In human heart sympathetic nerve stimulation via activation of β-adrenergic receptors activates calcium influx through the L-type calcium channel. It is known that cAMP-dependent protein kinase A (PKA) can phosphorylate the pore forming and voltage sensing α-subunit (Cav1.2) of the heterotetramer channel. In previous studies we showed that the N and C terminal regions do not contain the critical sites for PKA phosphorylation. There are 89 serine and 75 threonine residues in total along the C-truncated short N terminal isoform, but only 21 serines and 15 threonines positioned along the intracellular loops between repeats. In vitro phosphorylation of the Repeat I-II and Repeat II-III loop regions produced as GST-fusion peptides was performed and immunoblot technique was used to determine the PKA phosphorylation of purified channel proteins with PKA substrate specific antibody and phosphoprotein specific fluorescent labelling. The Repeat II-III region demonstrated increased phosphorylation (n=3). We mutated four serine residues to alanine at S436, S754, S834, and S1458 by site directed mutagenesis on the C truncated short terminal isoform. In vitro phosphorylation studies demonstrated a more pronounced increase in the phosphorylation level of the non-mutant protein vs mutant protein (n=3). We are now performing functional assessment of the single channel activity of the mutated serine containing channel protein reconstituted in proteoliposomes using patch-clamp technique. In conclusion our results suggest that the most likely candidates for PKA phosphorylation are the serine residues in the intracellular loop regions of Cav1.2 subunit.