Small conductance Ca²⁺-activated K⁺ channels and calmodulin. Cell surface expression and gating

Small conductance Ca²⁺-activated K⁺ channels (SK channels) are heteromeric complexes of pore-forming α subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CAM1,2,3,4 a mutant lacking the ability to bind Ca²⁺, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca²⁺-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca²⁺-free solution and were not reactivated upon reapplication of Ca²⁺-containing solution. Channel activity was restored by application of purified recombinant Ca²⁺-CaM or exposure to Ca²⁺-free CaM followed by application of Ca²⁺-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca²⁺-dependent channel activity that was not lost with exposure to Ca²⁺-free solution. Therefore, Ca²⁺-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.