Elucidating the Molecular Mechanisms for Activation of the L-Type Calcium Channel in the Fight or Flight Response

Abstract

In cardiac muscle sympathetic stimulation leads to an increase in heart rate and contraction as a result of increased calcium influx through the L-type Ca2+ channel (Cav1.2). This involves activation of the β-adrenergic receptor and an increase in cAMP dependent protein kinase A (PKA). It is unknown whether direct modification of Cav1.2 is responsible for altered channel function and as a result the mechanism has remained controversial. We searched for the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca2+ channel pore forming α1 subunit using a novel approach. Functional studies were performed on proteoliposomes containing human Cav1.2 channel protein and phosphatidylcholine lipid only. Using this approach channel function is studied in isolation from the auxiliary subunits of the channel complex and the regulatory pathways present in the cell. We confirm a direct effect of PKA on Cav1.2 channel function. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and a protein kinase A anchoring protein (AKAP) was not required. Mutation of Ser1928 and potential PKA substrate serines in the cytoplasmic Repeat I-II and II-III linker regions did not attenuate the effect nor truncation of the C terminus. We find that direct phosphorylation of a single serine that lies in close proximity to the Repeat IV S6 region on Cav1.2 alters posttranslational folding and is necessary and sufficient for an increase in function responsible for the “Fight or Flight” response.