Subtype‐selective positive modulation of KCa2 channels depends on the HA/HB helices

Background and Purpose In the activated state of small‐conductance Ca2+‐activated potassium (KCa2) channels, calmodulin interacts with the HA/HB helices and the S4‐S5 linker. CyPPA potentiates KCa2.2a and KCa2.3 channel activity but not the KCa2.1 and KCa3.1 subtypes. Experimental Approach Site‐directed mutagenesis, patch‐clamp recordings and in silico modelling were utilised to explore the structural determinants for the subtype‐selective modulation of KCa2 channels by CyPPA. Key Results Mutating residues in the HA (V420) and HB (K467) helices of KCa2.2a channels to their equivalent residues in KCa3.1 channels diminished the potency of CyPPA. CyPPA elicited prominent responses on mutant KCa3.1 channels with an arginine residue in the HB helix substituted for its equivalent lysine residue in the KCa2.2a channels (R355K). KCa2.1 channels harbouring a three‐amino‐acid insertion upstream of the cognate R438 residues in the HB helix showed no response to CyPPA, whereas the deletion mutant (KCa2.1_ΔA434/Q435/K436) became sensitive to CyPPA. In molecular dynamics simulations, CyPPA docked between calmodulin C‐lobe and the HA/HB helices widens the cytoplasmic gate of KCa2.2a channels. Conclusion and Implications Selectivity of CyPPA among KCa2 and KCa3.1 channel subtypes relies on the HA/HB helices.