title: Local anesthetic block of Kv channels: role of the S6 helix and the S5-S6 linker for bupivacaine action.
abstract: To gain insights in the molecular mechanisms of anesthesia, we analyzed the effects of bupivacaine on a series of voltage-gated K+ channels (Kv1.1, -1.2, -1.5, -2.1, -3.1, and -3.2) and various mutant channels derived from Kv2.1, using Xenopus laevis oocytes. Two phenomenologically different blocking effects were seen at room temperature: a time-dependent block of Kv1 and Kv3 channels (Kd between 110 and 240 microM), and a time-independent block on Kv2.1 (Kd = 220 microM). At 32 degrees C, however, Kv2.1 also showed a time-dependent block. Swapping the S6 helix between Kv1.2 and Kv2.1 introduced Kv1.2 features in Kv2.1. Critical residues were located in the N-terminal end of S6, positions 395 and 398. The triple substitution of residues 372, 373, and 374 in the S5-S6 linker decreased the bupivacaine affinity by 5-fold (Kd increased from 220 to 1170 microM). The results suggest that bupivacaine blocks Kv channels by an open-state-dependent mechanism and that Kv2.1 deviates from the other channels in allowing a partial closure of the channel with bupivacaine bound. The results also suggest that the binding site is located in the internal vestibule and that residues in the descending P-loop and the upper part of S6 are critical for the binding, most likely by allosteric mechanisms. A simple mechanistic scenario that explains the observations is presented. Thermodynamic considerations suggest that the interaction between bupivacaine and the channels is hydrophobic.
authors.affiliation: The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
authors.forename: Johanna
authors.initials: J
authors.lastname: Nilsson
authors.forename: Michael
authors.initials: M
authors.lastname: Madeja
authors.forename: Peter
authors.initials: P
authors.lastname: Arhem
mesh_terms: D000595:Amino Acid Sequence; D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002045:Bupivacaine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D000779:Anesthetics, Local; D015221:Potassium Channels; D002045:Bupivacaine