Phosphorylation sites in the Hook domain of CaVβ subunits differentially modulate CaV1.2 channel function

Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. Ca(V)1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC-MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of Ca-V beta subunits in vivo and test the impact of mutations of those sites on Ca(V)1.2 channel function in vitro. Using the Ca(V)1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser(193) and Thr(205) in the HOOK domain of Ca-V beta(1a) a subunits were both phosphorylated in vivo. Ser(193) is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr205 is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple Ca-V beta subunit isoforms, including the principal Ca-V beta subunit of cardiac Ca(V)1.2 channels, Ca-V beta(2b). In order to assess potential modulatory effects of phosphorylation at these sites separately from the effects of phosphorylation of the alpha(1)1.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in Ca-V beta(2b) and analyzed their effects on Ca(V)1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation Ca-V beta(S152E)(2b) decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation Ca-V beta(S152A)(2b) had opposite effects. There were no differences in peak Ca(V)1.2 currentsor voltage dependence between the phosphomimetic mutation Ca-V beta(T164D)(2b) and the phosphoinhibitory mutation Ca-V beta(T164A)(2b). However, calcium-dependent inactivation was significantly increased for the phosphomimetic mutation Ca-V beta(T164D)(2b). This effect was subunit-specific, as the corresponding mutation in the palmitoylated isoform, Cav132a, had no effect. Overall, our data identify two conserved sites of phosphorylation of the Hook domain of Ca-V beta subunits in vivo and reveal differential modulatory effects of phosphomimetic mutations in these sites. These results reveal a new dimension of regulation of Ca(V)1.2 channels through phosphorylation of the Hook domains of their beta subunits. (C) 2015 Elsevier Ltd. All rights reserved.