Homologous Serine/Threonine in the CaV 2.2α1 and 2.3α1 Subunits Behave Similarly, as Stimulatory and Inhibitory PKC Sites

Abstract

High voltage-gated calcium (Cav) channels are regulated by PKC isozymes. These isozymes target selected serine/threonine (Ser/Thr) PKC phosphorylation sites in the intracellular regions of Cavα1 subunits of these channels. It has been found earlier using Cav2.2α1 subunits that stimulatory (Thr-422, Ser-2108 and Ser-2132) and inhibitory (Ser-425) PKC sites exist and their activation with PKC isozymes led to potentiation and depression of calcium currents (ICa) respectively. Based on the above report, it was planned to examine if the homologous sites in the Cav2.3α1 subunits behave similarly. In this regard the WT Cav2.3α1 or Ser/Thr Ala mutants of stimulatory (Thr-365, Ser-1995 and Ser-2011) or inhibitory (Ser-369) sites were expressed along with β1b and 2/δ cDNA subunits in Xenopus oocytes and the barium currents (IBa) were studied. Intracellular injection of PKC isozymes βII or e potentiated WT Cav2.3 currents. While both PKC βII and e potentiated IBa through Thr-365 (T365/S369A/S1995A/S2011A), only PKC e increased IBa through Ser-1995 (T365A/S369A/S1995/S2011A) channels. Ser-2011 failed to act as a stimulatory site contrary to its homologous site, Ser-2132 in the Cav2.2α1 subunits. However, Ser-369 acted as inhibitory site as its homolog Ser-425 in the Cav2.2α1 subunits. Both PKC βII and e inhibited IBa through Ser-369 (T365A/S369/S1995A/S2011A) channels. When both Thr-365 and Ser-369 were present (T365/S369/S1995A/S2011A), IBa was neither stimulated nor inhibited. However, stimulation was dominant when two stimulatory sites (Thr-365 & Ser-1995) were present along with Ser-369. Experiments with other mutants, including Ser/Thr Asp constructs are being studied and will be discussed.