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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Allosteric modulation of a neuronal K⁺ channel by 1-alkanols is linked to a key residue in the activation gate

Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Submitted 27 March 2003 ; accepted in final form 27 May 2003

The selective inhibition of neuronal Shaw2 K⁺ channels by 1-alkanols is conferred by the internal S4-S5 loop, a region that also contributes to the gating of voltage-gated K⁺ channels. Here, we applied alanine scanning mutagenesis to examine the contribution of the S5 and S6 segments to the allosteric modulation of Shaw2 K⁺ channels by 1-alkanols. The internal section of S6 is the main activation gate of K⁺ channels. While several mutations in S5 and S6 modulated the inhibition of the channels by 1-butanol and others had no effect, a single mutation at a key site in S6 (P410A) converted this inhibition into a dramatic dose-dependent potentiation (2-fold at 15 mM and 6-fold at 50 mM). P410 is the second proline in the highly conserved PVP motif that may cause a significant -helix kink. The P410A currents in the presence of 1-butanol also exhibited novel kinetics (faster activation and slow inactivation). Internal application of 15 mM 1-butanol to inside-out patches expressing P410A did not significantly affect the mean unitary currents (2 pA at 0 mV) or the mean open time (5-6 ms) but clearly increased the opening frequency and open probability (2- to 4-fold). All effects displayed a fast onset and were fully reversible upon washout. The results suggest that the allosteric modulation of the Shaw2 K⁺ channel by 1-alkanols depends on a critical link between the PVP motif and activation gating. This study establishes the Shaw2 K⁺ channel as a robust model to investigate the mechanisms of alcohol intoxication and general anesthesia.

alcohol; anesthesia; gating; scanning mutagenesis; Shaw channels

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