Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca2+ sensitivity of SK2 channels

Aim: Small-conductance Ca2+-activated potassium (SK) channels are activated exclusively by increases in intracellular Ca2+ that binds to calmodulin constitutively associated with the channel. Wild-type SK2 channels are activated by Ca2+ with an EC50 value of similar to 0.3 mu mol/L. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca(2+)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(2+)hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca2+. The Ca(2+)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(2+)sensitivity. The double N293A/V407F mutations generate a highly Ca(2+)sensitive channel, with an EC50 of 0.02 mu mol/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 Ca2+ sensitivity of SK2 channels.

Automated summary

The hydrophobic interactions between the HA helix and the S4-S5 linker are found to be the main determinant of channel apparent Ca(2+) sensitivity in SK2 channels. Mutations that alter the hydrophobicity at this interface can lead to either hypersensitivity or hyposensitivity to Ca2+. This study highlights the crucial role of interactions between the HA helix and S4-S5 linker in regulating the Ca(2+) sensitivity of SK2 channels.