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 coass embled complexes of pore-forming SK alpha subunits and calmodulin. We propo sed a model for channel activation in which Ca2+ binding to calmodulin indu ces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that ind icate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experi ments showed that the Ca2+-independent interaction of calmodulin with the a lpha subunits requires only the C-terminal domain of calmodulin and is medi ated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmoduli n-binding motif, mutagenesis experiments identified two positively charged residues required for Ca2+-independent interactions with calmodulin. Electr ophysiological 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 dom ain of calmodulin. Taken together, the results support a calmodulin- and Ca 2+-calmodulin-dependent conformational change in the channel alpha subunits , in which different domains of calmodulin are responsible for Ca2+-depende nt and Ca2+-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel ga ting. Based on these results, a state model for Ca2+ gating was developed t hat simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.