Reconstitution of PKA-Dependent Modulation of Cardiac Cav1.2 Channels

Abstract

L-type calcium currents through Cav1.2 channels initiate contraction in cardiac muscle. Their regulation by neurotransmitters and hormones through second messenger signaling cascades and protein kinase A (PKA) phosphorylation is a key controller of calcium signaling and contractile force. The α1 subunit C-terminus contains binding sites for multiple regulatory proteins including the PKA/A kinase anchoring protein 15 (AKAP15) complex. The C-terminal domain is proteolytically cleaved but reassociates non-covalently with the truncated channel and acts as a potent autoinhibitor of channel activity. Relief of autoinhibition by cellular regulatory signals acting on the C-terminus provides an attractive mechanism for producing the large increases in calcium current that are observed physiologically. In fact, consistent reconstitution of PKA-dependent regulation of Cav1.2 channels in non-muscle has been difficult to achieve. To reconstitute such PKA regulation, we optimized the expression of truncated Cav1.2 channels, the distal C-terminal domain, the α2δ subunit, and the β2b subunit to give a functional autoregulatory complex as assessed by whole-cell voltage clamp recordings of tsA-201 cells. Expression of the truncated Cav1.2 channel with the free distal domain resulted in large decreases in inward barium current and in coupling between voltage-dependent gating and pore opening. We hypothesized that AKAP15 expression would promote PKA association with the distal C-terminal of the channel and increase the likelihood of PKA-dependent phosphorylation. After optimizing AKAP15 expression, currents recorded in 5 μM forskolin were approximately 5-fold larger than those recorded in the presence of kinase inhibitor RO 31-8220 (1 uM). These findings show that the full-range of PKA-dependent modulation of Cav1.2 channels can be reproduced when an autoinhibitory complex is formed in this manner and provide a substrate for further studies of this physiologically important regulatory process.Supported by NIH grant HL007312-31 and AHA fellowship 09POST2080270.