Kinase-dependent phosphorylation of the intracellular proximal C terminus is needed for VI Cav2.3 channel inhibition by c-Vc1.1. (A) Amino acid sequence alignments of e37 regions in splice variants of Cav2.2, Cav2.1, and Cav2.3 and mutant Cav2.1 and Cav2.3 genes. Y (red), followed by A, E, or T (orange), is shown as a possible substrate for c-Src phosphorylation. (Right) Proposed model for c-Vc1.1–induced intracellular signaling leading to Cav2 channel inhibition. Asterisk indicates phosphorylation. (B) Average IBa inhibition in the presence of 200 nM c-Vc1.1 or 50 µM baclofen in HEK 293T cells transiently cotransfected with wild-type human Cav2.3d (α1E-d) and Cav2.3c (α1E-c); Cav2.1 (α1A-5); rabbit Cav2.1 channel subunits; human e37 mutants Cav2.3d (Y1761F), Cav2.3d (Y1765F), Cav2.3c (Y1761F), Cav2.3c (Y1765F), Cav2.1 (α1A-5) (L1852T), or Cav2.1 (α1A-5) (Q1852E); auxiliary human Cav channel subunits α2bδ-1 and β3; and human GABABR subunits. Representative normalized IBa traces (current insets) of wild-type or mutant channels are shown in the absence (control) or presence of 200 nM c-Vc1.1 or 50 µM baclofen (only 65 ms of the 150-ms current traces are plotted). IBa was evoked by depolarizations to +10 mV (Cav2.3c) or 15 mV (Cav2.1), applied at 0.1 Hz from an HP of −80 mV. Dotted lines indicate zero-current levels. Bar graphs show mean ± SEM; n, number of experiments in parentheses. Labels a, b, and c denote statistically significant differences between various mutants and control; NS, not significantly different from wild-type modulation. See Table 3 for statistical evaluation of the data.
