The Concerted Contribution of the S4-S5 Linker and the S6 Segment to the Modulation of a Kv Channel by 1-Alkanols

Abstract

Gating of voltage-gated K⁺ channels (K_v channels) depends on the electromechanical coupling between the voltage sensor and activation gate. The main activation gate of K_v channels involves the COOH-terminal section of the S6 segment (S6-b) and the S4-S5 linker at the intracellular mouth of the pore. In this study, we have expanded our earlier work to probe the concerted contribution of these regions to the putative amphipathic 1-alkanol site in the Shaw2 K⁺ channel. In the S4-S5 linker, we found a direct energetic correlation between α-helical propensity and the inhibition of the Shaw2 channel by 1-butanol. Spectroscopic structural analyses of the S4-S5 linker supported this correlation. Furthermore, the analysis of chimeric Shaw2 and K_v3.4 channels that exchanged their corresponding S4-S5 linkers showed that the potentiation induced by 1-butanol depends on the combination of a single mutation in the S6 PVPV motif (PVAV) and the presence of the Shaw2 S4-S5 linker. Then, using tandem-heterodimer subunits, we determined that this potentiation also depends on the number of S4-S5 linkers and PVAV mutations in the K_v channel tetramer. Consistent with the critical contribution of the Shaw2 S4-S5 linker, the equivalent PVAV mutation in certain mammalian K_v channels with divergent S4-S5 linkers conferred weak potentiation by 1-butanol. Overall, these results suggest that 1-alkanol action in Shaw2 channels depends on interactions involving the S4-S5 linker and the S6-b segment. Therefore, we propose that amphiphilic general anesthetic agents such as 1-alkanols may modulate gating of the Shaw2 K⁺ channel by an interaction with its activation gate.