Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca sensitivity of SK2 channels.

Aim: Small-conductance Ca -activated potassium (SK) channels are activated exclusively by increases in intracellular Ca that binds to calmodulin constitutively associated with the channel. Wild-type SK2 channels are activated by Ca with an EC value of ~0.3 μmol/L. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca sensitivity.

Methods: Site-directed mutagenesis, electrophysiological recordings and molecular dynamic (MD) simulations were utilized.

Results: Mutations that decrease hydrophobicity at the HA-S4-S5 interface lead to Ca hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca . The Ca hypersensitivity of the V407F mutant relies on the interaction of the cognate phenylalanine with the S4-S5 linker in the SK2 channel. Replacing the S4-S5 linker of the SK2 channel with the S4-S5 linker of the SK4 channel results in loss of the hypersensitivity caused by V407F. This difference between the S4-S5 linkers of SK2 and SK4 channels can be partially attributed to I295 equivalent to a valine in the SK4 channel. A N293A mutation in the S4-S5 linker also increases hydrophobicity at the HA-S4-S5 interface and elevates the channel apparent Ca sensitivity. The double N293A/V407F mutations generate a highly Ca sensitive channel, with an EC of 0.02 μmol/L. The MD simulations of this double-mutant channel revealed a larger channel cytoplasmic gate.

Conclusion: The electrophysiological data and MD simulations collectively suggest a crucial role of the interactions between the HA helix and S4-S5 linker in the apparent Ca sensitivity of SK2 channels.