Calmodulin mediates calcium-dependent activation of the intermediate conductance K-Ca channel, IKCa1

Small and intermediate conductance Ca2+-activated K+ channels play a crucia l role in hyperpolarizing the membrane potential of excitable and nonexcita ble cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, ye t their protein-coding regions do not contain consensus Ca2+-binding motifs . me investigated the involvement of an accessory protein in the Ca2+-depen dent gating of hIKCa1, a human intermediate conductance channel expressed i n peripheral tissues. Calmodulin was found to interact strongly with the cy toplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletio n analyses defined a requirement for the first 62 amino acids of the C-tail , and the binding of calmodulin to this region did not require Ca2+. The C- tail of hSKCa3, a human neuronal small conductance channel, also bound calm odulin, whereas that of a voltage-gated K+ channel, mKv1.3, did not. Calmod ulin coprecipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1.3-transfected Lines. A mutant calmodulin, defective in C a2+ sensing but retaining binding to the channel, dramatically reduced curr ent amplitudes when co-expressed with hIKCa1 in mammalian cells. Co-express ion with varying amounts of wild-type and mutant calmodulin resulted in a d ominant-negative suppression of current, consistent with four calmodulin mo lecules being associated with the channel. Taken together, our results sugg est that Ca2+ calmodulin-induced conformational changes in all four subunit s are necessary for the channel to open.