FIGURE 3. Three acidic amino acid residues, Glu-1122, Asp-1127 and Asp-1129, positioned proximal to the pore helix in domain III are important for calcicludine binding to Ca_V1.2 channels.

Wild-type, Mut-A, Mut-B and Mut-C (A) and Wild-type, E1122A, D1127A and D1129A (B) Ca²⁺ channels were incubated with [³H]PN200-110 and the indicated concentrations of calcicludine as described in the Materials and Methods. Data were fit using Scheme 1 (see Equation 4, Materials and Methods) and normalized such that the occupancy in zero calcicludine is equal to 1.0. The method for fitting binding data from the individual mutants, E1122A, D1127A and D1129A, is described in the text and Legend for Table 1. DHP binding to Mut-A and Mut-C membranes does not increase in the presence of calcicludine, but block of Mut-A and not Mut-C currents by calcicludine is similar to that of wild-type (Fig. 5; see text for details). Data are means ± SEM, and significant differences between binding parameters of wild-type and mutant channels were evaluated using a 2-tailed Students-t test P<0.05 (*). Error bars smaller than symbols do not appear in figures. See Table 1 for data summary and indications of statistical significance. (A) Wild-type (n = 6), Mut-A (n = 3), Mut-B (n = 3), Mut-C (n = 3); (B) Wild-type (n = 6), E1122A (n = 3), D1127A (n = 3), D1129A (n = 3).