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

TLDR

Small and intermediate conductance Ca²⁺‑activated K⁺ channels hyperpolarize cell membranes and are highly sensitive to cytoplasmic Ca²⁺, yet their coding sequences lack canonical Ca²⁺‑binding motifs. The study aimed to determine whether an accessory protein mediates Ca²⁺‑dependent gating of the human intermediate conductance channel hIKCa1. Using yeast two‑hybrid assays, deletion mapping, co‑precipitation, and functional expression with mutant calmodulin, the authors investigated calmodulin’s interaction with the channel’s C‑terminal tail. Calmodulin binds the first 62 amino acids of hIKCa1’s C‑tail independently of Ca²⁺, and a Ca²⁺‑sensing‑defective calmodulin mutant markedly suppresses channel currents, indicating that Ca²⁺‑induced conformational changes in all four calmodulin molecules are required for channel opening.

Abstract

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.