Molecular Mechanism of Calcium Channel Regulation in the Fight-Or-Flight Response

Abstract

During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium currents in the heart conducted by Cav1.2 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by cAMP-dependent protein kinase (PKA), thereby increasing cardiac contractility and beat rate. The channel α1 subunit C-terminus contains binding sites for multiple regulatory proteins including the PKA/A kinase anchoring protein 15 (AKAP15) complex. The C-terminus is proteolytically cleaved but remains associated non-covalently with the truncated channel and acts as a potent autoinhibitor of channel activity. Relief of this autoinhibition provides an attractive mechanism for cellular regulatory signals to produce the large increases in calcium current observed physiologically. We reconstituted regulation of Cav1.2 channels in non-muscle tsA-201 cells by forming an autoregulatory signaling complex composed of the Cav1.2Δ1800 channel, the noncovalently-associated distal C-terminal domain, the auxiliary α2δ1 and β2b subunits, and AKAP15. During whole-cell recordings of channel activity we observed a 3.6-fold range of Cav1.2 activity from a minimum in the presence of protein kinase inhibitors to a maximum with activation of adenylyl cyclase with forskolin. Equivalent modulation was not observed for the full-length (untruncated) channel or the truncated channel without the distal C-terminus. Basal channel activity in unstimulated cells was regulated by phosphorylation of two novel sites at Ser1700 in a PKA consensus sequence and Thr1704 in a casein kinase 2 consensus sequence, both strategically located at the interface between the distal and proximal C-terminal regulatory domains. Further stimulation of channel activity via PKA signaling required only phosphorylation of Ser1700. Phosphorylation at Ser1928 did not significantly alter channel activity. These results define the signaling complex required for Cav1.2 channel regulation and elucidate the sites of phosphorylation that regulate channel activity.Supported by NIH grants R01 HL085372, T32 HL007312-31 and AHA fellowship 09POST2080270