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

Abstract

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 ~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 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 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 Ca²⁺ sensitivity of SK2 channels.

Keywords: HA helix; S4-S5 linker; SK2 channels.

Fig. 1.. Alanine substitution of the hydrophobic residues in the S4–S5 linker causes hyposensitivity of SK2 channels to Ca²⁺.

(a) A homology model of the rat SK2 channel was generated using the human SK4 channel cryo-EM structure with Ca²⁺. (b) Ca²⁺ bound CaM (blue), HA and HB helices from one channel subunit (green), the S₄₅A and S₄₅B helices from a neighboring channel subunit (yellow) become a stable structural group right at the bottom of the pore-forming S6 transmembrane domain. (c) Concentration-dependent activation by Ca²⁺ of the WT and alanine mutant SK2 channels. (d) EC₅₀ values for activation by Ca²⁺ of the WT and alanine mutant channels. ‡ P < 0.001 compared with WT. No asterisk means no statistical significance compared with WT.

Fig. 2.. Mutations of N293 in the S₄₅A helix change apparent Ca²⁺ sensitivity of SK2 channels.

(a) Amino acid sequence alignment of human SK1[GenBank: NP_002239.2], rat SK2 [Genbank: NP_062187.1], human SK3 [GenBank: NP_002240.3] and human SK4 [GenBank: NP_002241.1] at the S₄₅A and S₄₅B helices (highlighted in yellow) and the proximal end of HA helix (highlighted in green). (b) Activation by Ca²⁺ of the WT and channels carrying mutations at N293. (c) EC₅₀ values for activation by Ca²⁺ of the WT and mutant channels. * P < 0.05, ‡ P < 0.001 compared with WT. No asterisk means no statistical significance compared with WT.

Fig. 3.. Mutations of asparagine in S₄₅A helix changes both SK2 and SK4 apparent Ca²⁺ sensitivity, while mutations of the valine in HA helix increases SK2 but not SK4 apparent Ca²⁺ sensitivity.

(a) Concentration-dependent activation of the WT and N-to-A mutant SK2 and SK4 current by Ca²⁺. (b) EC₅₀ values for the activation of the WT and N-to-A mutant SK channels by Ca²⁺. (c) Concentration-dependent activation of the WT and V-to-F mutant SK2 and SK4 current by Ca²⁺. (d) EC₅₀ values for the activation of the WT and V-to-F mutant SK channels by Ca²⁺. ‡ P < 0.001 compared with respective WT channel subtype. No asterisk means no statistical significance compared with respective WT channel subtype.

Fig. 4.. The introduction of V298F mutation leads to Ca²⁺ hypersensitivity of the SK4__S4-S5-SK2 chimera.

(a) A chimeric SK4 channel carrying the S4–S5 linker of SK2 channels (SK4__S4-S5-SK2), and a chimeric SK2 channel carrying the S4–S5 linker of SK4 channels (SK2__S4-S5-SK4) were generated. The S4–S5 linkers swapped between the two subtypes are shown in bold. The S4–S5 linker from SK2 channel is shown in black, while that of the SK4 channel is shown in blue font. Two critical differences in the S4–S5 linker between the subtypes are shown in red font. (b) Introduction of V298F mutation left-shifts the concentration-dependent activation of the SK4__S4-S5-SK2 chimeric but not the WT SK4 channel. (c) EC₅₀ values for the activation of the chimeric SK4 channels by Ca²⁺. ‡ P < 0.001. No asterisk means no statistical significance.

Fig. 5.. The introduction of V407F mutation fails to increase apparent Ca²⁺ sensitivity of the SK2__S4-S5-SK4 chimera.

(a) Introduction of V407F mutation left-shifts the concentration-dependent activation of the WT SK2 channels, but right-shifts the SK2__S4-S5-SK4 chimeric channel. (b) EC₅₀ values for the activation of the chimeric SK2 channels by Ca²⁺. ‡ P < 0.001.

Fig. 6.. An isoleucine I295 residue in the S4–S5 linker is required for V407F to induce Ca²⁺ hypersensitivity in SK2 channels.

(a) An isoleucine (I295) from the S₄₅A helix (yellow)is in the vicinity of V407 from the HA helix (green), while a serine (S286) is beyond 5 angstroms. (b) I295V mutation right-shifts the Ca²⁺ dependent activation of the V407F mutant SK2 channel. (c) I295V mutation significantly compromised the Ca²⁺ hypersensitivity of the V407F mutant SK2 channel. † P < 0.01, ‡ P < 0.001. No asterisk means no statistical significance.

Fig. 7.. N293A and V407F mutations additively increase apparent Ca²⁺ sensitivity in SK2 channels.

(a) N293A/V407F double mutations further left-shifted the Ca²⁺ dependent activation of the SK2 channel. (b) N293A/V407F double mutations increase the apparent Ca²⁺ sensitivity of SK2 channels to ~0.02 μM. † P < 0.01, ‡ P < 0.001. (c) In MD simulations, the minimum distance at the cytoplasmic gate is shown as a function of the simulation time. (d) The distribution histogram of the minimum distance at the cytoplasmic gate shows a larger distance in the N293A/V407F double mutant channel.