Clarifying a Novel PKA Phosphorylation Site on the L-type Calcium Channel

Abstract

Persistent sympathetic activation leads to the development of hypertrophy and heart failure. Activation of the L-type calcium channel is essential for the increase in contractility and heart rate however the mechanism for the increase in channel function has remained controversial for over 40 years. The downstream effector of β-adrenergic signalling pathway is the cyclic AMP-dependent protein kinase A (PKA). We recently identified a novel candidate PKA phosphorylation site on Cav1.2 protein (the main pore forming and ion conducting alpha subunit). We reconstituted the purified channel isoforms in liposomes. Using single channel patch-clamp analysis, mutation of serine 1458 to alanine attenuated the effect of PKA on channel function (Po = 0.052 ± 0.01 vs 0.043 ± 0.007, n = 5; p = NS). In vitro thermal stability assay performed on the S1458A mutant demonstrated similar melting peak profiles. Following 1 hr in vitro PKA treatment the melting temperature of the WT isoform was altered, but not the mutant isoform, demonstrating that protein-protein interactions between PKA and the Cav1.2 had occurred. Mass spectrometry analysis was performed to demonstrate that serine 1458 could be phosphorylated by PKA. We performed in vitro PKA phosphorylation of the non-phosphorylated synthetic peptide YLTRDWSILGPHHL, corresponding to aa 1452-1465 of the short NT isoform then performed MS/MS analysis. Peptide ions for YLTRDWSILGPHHL were observed with a mass of 1706.86 in control and 1786.84 in PKA treated samples, and we could track the mass difference to the serine residue in the MS/MS spectra. In conclusion, we identified a novel, PKA specific phosphorylation site on the proximal C terminus of Cav1.2 protein.