Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance KCa Channel,IKCa1

Small and intermediate conductance Ca²⁺-activated K⁺ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca²⁺, yet their protein-coding regions do not contain consensus Ca²⁺-binding motifs. We investigated the involvement of an accessory protein in the Ca²⁺-dependent gating of <i>hIKCa1</i>, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of <i>hIKCa1</i> in a yeast two-hybrid system. Deletion 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<i>hSKCa3,</i> a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K⁺channel, <i>mKv1.3,</i> did not. Calmodulin co-precipitated with the channel in cell lines transfected with <i>hIKCa1,</i> but not with <i>mKv1.3</i>-transfected lines. A mutant calmodulin, defective in Ca²⁺ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed with<i>hIKCa1</i> in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca²⁺-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.