Domains responsible for constitutive and Ca²⁺-dependent interactions between calmodulin and small conductance Ca²⁺-activated potassium channels

Small conductance Ca²⁺-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK a subunits and calmodulin. We proposed a model for channel activation in which Ca²⁺ binding to calmodulin induces conformational rearrangements in calmodulin and the a subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the a subunit after calmodulin binding and Ca²⁺ binding to the α subunit-calmodulin complex. Two-hybrid experiments showed that the Ca²⁺-independent interaction of calmodulin with the subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca²⁺ is not required. Although SK α subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca²⁺-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca²⁺ binding to the first and second E-F hand motifs in the N- terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca²⁺calmodulin-dependent conformational change in the channel α subunits, in which different domains of calmodulin are responsible for Ca²⁺-dependent and Ca²⁺-independent interactions. In addition, calmodulin is associated with each α subunit and must bind at least one Ca²⁺ ion for channel gating. Based on these results, a state model for Ca²⁺ gating was developed that simulates alterations in SK channel Ca²⁺ sensitivity and cooperativity associated with mutations in CaM.