Received for publication May 2, 2006.
Revised August 3, 2006.
Accepted for publication August 3, 2006.

THE CONCERTED CONTRIBUTION OF THE S4-S5 LINKER AND THE S6 SEGMENT TO THE MODULATION OF A K_v CHANNEL BY 1-ALKANOLS

Abstract

Gating of voltage-gated K⁺ channels (Kv channels) depends on the electromechanical coupling between their voltage sensor and activation gate. The main activation gate of Kv channels involves the COOH-terminal section of the S6 segment (S6-b) and the S4-S5 linker at the intracellular mouth of the pore. Here, we have expanded our earlier studies 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 -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 Kv3.4 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 Kv channel tetramer. Consistent with the critical contribution of the Shaw2 S4-S5 linker, the equivalent PVAV mutation in certain mammalian Kv 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.

Key words: Ion channel regulation, Potassium, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, NMR, Alcohol

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