Positive modulation of SKs channels by CyPPA depends on the HA/HB helices

In the activated state of small-conductance Ca2+-activated potassium (SK) channels, calmodulin interacts with the HA/HB helices and the S4-S5 linker. CyPPA potentiates SK2a and SK3 channel activity but not the SK1 and IK subtypes. Here, we report that the subtype-selectivity of CyPPA relies on the HA/HB helices. Mutating residues in the HA (V420) and HB (K467) helices of SK2a channels to their equivalent residues in IK channels diminished the potency of CyPPA. CyPPA elicited prominent responses on mutant IK channels with an arginine residue in the HB helix substituted for its equivalent lysine residue in the SK2a channels (R355K). SK1 channels harboring a three-amino-acid insertion upstream of the cognate R438 residues in the HB helix showed no response to CyPPA, whereas the deletion mutant (SK1_ΔA434/Q435/K436) became sensitive to CyPPA. In molecular dynamics simulations, CyPPA docked between calmodulin and the HA/HB helices widens the cytoplasmic gate of SK2a channels.