title: Allosteric modulation of a neuronal K+ channel by 1-alkanols is linked to a key residue in the activation gate.
abstract: 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 (approximately 2-fold at 15 mM and approximately 6-fold at 50 mM). P410 is the second proline in the highly conserved PVP motif that may cause a significant alpha-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 (approximately 2 pA at 0 mV) or the mean open time (5-6 ms) but clearly increased the opening frequency and open probability (approximately 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.
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College of Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107, USA.
authors.forename: Thanawath
authors.initials: T
authors.lastname: Harris
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Graber
authors.forename: Manuel
authors.initials: M
authors.lastname: Covarrubias
mesh_terms: D020001:1-Butanol / Q000008:administration & dosage / Q000494:pharmacology*; D000494:Allosteric Regulation; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D015640:Ion Channel Gating; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D011392:Proline; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D014982:Xenopus laevis
chemical_list: D015221:Potassium Channels; D020001:1-Butanol; D011392:Proline