Ser1928 is Required for Regulation of Calcium-Dependent Inactivation of CaV1.2 L-Type Calcium Channels by AKAP79-Anchored PKA and Calcineurin

Protein kinase A (PKA) and the Ca2+/calmodulin-activated phosphatase calcineurin (CaN) reciprocally regulate CaV1.2 Ca2+ channel activity. The A-kinase anchoring protein AKAP79, associated with the C-terminus of CaV1.2 channels, scaffolds these opposing enzymes to the channel and, in neurons, thereby helps organize downstream signaling to the nucleus. Here we studied the role of phosphorylation signaling in CaN-mediated Ca2+-dependent inactivation (CDI) of CaV1.2 channels, focusing on sites in the channel's C-terminus that can be phosphorylated by PKA (Ser1700, Thr1704, Ser1928). We used voltage-clamp recording of Ca2+ or Ba2+ current carried by CaV1.2 channels (α11.2a, β2b, and α2δ1a subunits) transfected into tsA201 cells. Cells were also transfected with either wild-type or mutant AKAP79 constructs, and PKA activity was elevated with forskolin (FSK, 5 μM in whole-cell pipet). As previously reported, over-expression of an AKAP79 construct in which the CaN anchoring site was deleted (AKAP79ΔPIX) resulted in a decreased rate of CDI of FSK-enhanced Ca2+ current. Over-expression of the catalytically-inactive mutant CaNH151A also resulted in a decreased rate of CDI for FSK-enhanced current. A combination of CaN inhibitors, cyclosporin A and FK506, reversibly reduced the rate of CDI for FSK-enhanced current. Ser-to-Ala substitution at Ser1928 (S1928A) reduced the rate of CDI for Ca2+ current in 0 FSK, but the combination of FSK (perhaps stimulating PKA action at sites other than S1928) and over-expression of AKAP79ΔPIX overcame the effect of S1928A and restored CDI to the control rate. Other sites of phosphorylation by PKA in the CaV1.2 channel were investigated as well: the decrease in rate of CDI observed for wild-type channels co-expressed with AKAP79ΔPIX was present in the double mutant S1700A+T1704A. Our results suggest that S1928 is a key structural determinant in CDI of CaV1.2 channels.