Small conductance Ca 2ϩ -activated K ϩ channels (SK channels) are complexes of four ␣ pore-forming subunits each bound by calmodulin (CaM) that mediate Ca 2ϩ gating. Proteomic analysis indicated that SK2 channels also bind protein kinase CK2 (CK2) and protein phosphatase 2A (PP2A). Coexpression of SK2 with the CaM phosphorylation surrogate CaM(T80D) suggested that the apparent Ca 2ϩ sensitivity of SK2 channels is reduced by CK2 phosphorylation of SK2-bound CaM. By using

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... enzimidazole, a CK2-specific inhibitor, we confirmed that SK2 channels coassemble with CK2. PP2A also binds to SK2 channels and counterbalances the effects of CK2, as shown by coexpression of a dominant-negative mutant PP2A as well as a mutant SK2 channel no longer able to bind PP2A. In vitro binding studies have revealed interactions between the N and C termini of the channel subunits as well as interactions among CK2 ␣ and ␤ subunits, PP2A, and distinct domains of the channel. In the channel complex, lysine residue 121 within the N-terminal domain of the channel activates SK2-bound CK2, and phosphorylation of CaM is state dependent, occurring only when the channels are closed. The effects of CK2 and PP2A indicate that native SK2 channels are multiprotein complexes that contain constitutively associated CaM, both subunits of CK2, and at least two different subunits of PP2A. The results also show that the Ca 2ϩ sensitivity of SK2 channels is regulated in a dynamic manner, directly through CK2 and PP2A, and indirectly by Ca 2ϩ itself via the state dependence of CaM phosphorylation by CK2.