Mutant cycle analysis of K_D indicates that D50 and K61 residues are coupled in a Ca²⁺-sensitive manner. (A) Scheme for mutant cycle analysis of wild-type and mutant hemichannels. (B) Graph shows the [Ca²⁺] dose–response relations for oocytes expressing D50N (open circles) and K61R (crossed squares) mutants, and D50N/K61R (crossed circles) double mutant hemichannels. The solid and dotted lines represent the best fits to the Hill equation for wild-type (from Fig. 1 B) and mutant channels, respectively. (C) Deactivation time constants for oocytes expressing wild-type (closed circles), D50N (open circles), and K61R (crossed square) mutants, and D50N/K61R (fast and slow time constants are shown as two sets of crossed circles) double mutant hemichannels. The solid line corresponds to the best fits to the average data for wild-type hemichannels using Eq. 3. The dotted line is the best fits to the data to Eq. 3 for single and double mutant hemichannels.