ncreases 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 ~0.3 μM. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca2+ 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 Ca2+ hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca2+ . The Ca2+ hypersensitivity of the V407F mutant relies on the interact... More
ncreases 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 ~0.3 μM. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca2+ 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 Ca2+ hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca2+ . The Ca2+ 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 Ca2+ sensitivity. The double N293A/V407F mutations generate a highly Ca2+ sensitive channel, with an EC50 of 0.02 μM. 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.
