Channel activation. (A) Schematic drawing of the three polypeptides that coassemble in the A2-A2-A4-B1b channel, indicating the position of R/E mutations that were introduced to disable cAMP binding. The black sections represent the six transmembrane domains of each subunit, the cAMP-binding sites are depicted green, and the calmodulin-binding sites are shown in red. (B) I/V_m relations illustrating experiments designed to determine the cAMP sensitivity. The applied cAMP concentration is depicted to the right of each current trace. Selection parameters for A2-A2-A4-B1b channels were: (1) $K1/2−50$/$K1/250$ > 1.3; (2) K_1/2 different from 30 μM (A2-A2-A2-B1b) or 10 μM (A2-A2-A2-A4); and (3) Hill coefficient different from 1.8. (C) Contribution of subunits to cAMP sensitivity. The cAMP dose–response relation for wild-type A2-A2-A4-B1b channels was fitted with K_1/2 = 7.3 ± 1.7 μM, n = 1.71 ± 0.3 (black). Mutant channels yielded the following fit parameters: A2-A2-A4^R430E-B1b: K_1/2 = 37.6 ± 8.5 μM, n = 1.37 ± 0.2 (green); A2-A2-A4-B1b^R657E: K_1/2 = 25.5 ± 10.5 μM, n = 1.10 ± 0.1 (red); and A2-A2-A4^R430E-B1b^R657E: K_1/2 = 359.5 ± 68.5 μM, n = 1.28 ± 0.2 (blue).