Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca(2+) via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca(2+) and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca(2+)-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca(2+). These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca(2+)-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca(2+) affinity than WT CaM.