Small conductance Ca²⁺-activated K⁺ (SK) channels are heteromeric complexes composed of pore-forming α subunits and constitutively associated calmodulin (CaM), which binds to a calmodulin-binding domain (CaMBD) in the intracellular C terminus of the channel. Using electrophysiology and immunochemistry, we examined the SK2 double charge-reversal mutant (SK2 R464E/K467E), which exhibits markedly reduced CaM binding due to disruption of key salt bridges with CaM. These studies demonstrate that Ca²⁺-independent CaM association is essential for SK channel surface expression but not for channel gating. Site-directed mutagenesis and fluorescence labeling further identified critical hydrophobic and electrostatic interactions between the CaMBD and the C-lobe of CaM. Modification of SK2 L457C with methanethiosulfonate reagents revealed charge-dependent modulation of Ca²⁺ sensitivity, while crosslinking restored Ca²⁺ sensitivity. Together, these findings indicate that both electrostatic and hydrophobic interactions tightly couple CaM to the SK channel CaMBD, regulating channel expression and Ca²⁺ responsiveness.
