title: Ion selectivity filter regulates local anesthetic inhibition of G-protein-gated inwardly rectifying K+ channels.
abstract: The weaver mutation (G156S) in G-protein-gated inwardly rectifying K+ (GIRK) channels alters ion selectivity and reveals sensitivity to inhibition by a charged local anesthetic, QX-314, applied extracellularly. In this paper, disrupting the ion selectivity in another GIRK channel, chimera I1G1(M), generates a GIRK channel that is also inhibited by extracellular local anesthetics. I1G1(M) is a chimera of IRK1 (G-protein-insensitive) and GIRK1 and contains the hydrophobic domains (M1-pore-loop-M2) of GIRK1 (G1(M)) with the N- and C-terminal domains of IRK1 (I1). The local anesthetic binding site in I1G1(M) is indistinguishable from that in GIRK2(wv) channels. Whereas chimera I1G1(M) loses K+ selectivity, although there are no mutations in the pore-loop complex, chimera I1G2(M), which contains the hydrophobic domain from GIRK2, exhibits normal K+ selectivity. Mutation of two amino acids that are unique in the pore-loop complex of GIRK1 (F137S and A143T) restores K+ selectivity and eliminates the inhibition by extracellular local anesthetics, suggesting that the pore-loop complex prevents QX-314 from reaching the intrapore site. Alanine mutations in the extracellular half of the M2 transmembrane domain alter QX-314 inhibition, indicating the M2 forms part of the intrapore binding site. Finally, the inhibition of G-protein-activated currents by intracellular QX-314 appears to be different from that observed in nonselective GIRK channels. The results suggest that inward rectifiers contain an intrapore-binding site for local anesthetic that is normally inaccessible from extracellular charged local anesthetics.
authors.affiliation: The Salk Institute for Biological Studies, Peptide Biology Lab, La Jolla, California 92037, USA. slesinger@salk.edu
authors.forename: P A
authors.initials: PA
authors.lastname: Slesinger
mesh_terms: D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008012:Lidocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
chemical_list: D000779:Anesthetics, Local; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; C012647:QX-314; D001464:Barium; D008012:Lidocaine; D019204:GTP-Binding Proteins