title: Determinants of the anesthetic sensitivity of two-pore domain acid-sensitive potassium channels: molecular cloning of an anesthetic-activated potassium channel from Lymnaea stagnalis.
abstract: Certain two-pore domain K(+) channels are plausible targets for volatile general anesthetics, yet little is known at the molecular level about how these simple agents cause channel activation. The first anesthetic-activated K(+) current I(K(An)) that was characterized was discovered in the mollusk Lymnaea stagnalis and is remarkable for both its sensitivity to general anesthetics and its stereoselective responses to anesthetic enantiomers (Franks, N. P., and Lieb, W. R. (1988) Nature 333, 662-664 and Franks, N. P., and Lieb, W. R. (1991) Science 254, 427-430). Here we report the molecular cloning of a two-pore domain K(+) channel LyTASK from L. stagnalis and show that, when expressed in HEK-293 cells, it displays the same biophysical characteristics as the anesthetic-activated K(+) current I(K(An)). Sequence analysis and functional properties show it to be a member of the TASK family of channels with approximately 47% identity at the amino acid level when compared with human TASK-1 and TASK-3. By using chimeric channel constructs and site-directed mutagenesis we have identified the specific amino acid 159 to be a critical determinant of anesthetic sensitivity, which, when mutated to alanine, essentially eliminates anesthetic activation in the human channels and greatly reduces activation in LyTASK. The L159A mutation in LyTASK disrupts the stereoselective response to isoflurane while having no effect on the pH sensitivity of the channel, suggesting this critical amino acid may form part of an anesthetic binding site.
authors.affiliation: Biophysics Section, Blackett Laboratory, and Division of Biology, Imperial College, South Kensington, London SW7 2AZ.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Andres-Enguix
authors.forename: Alex
authors.initials: A
authors.lastname: Caley
authors.forename: Raquel
authors.initials: R
authors.lastname: Yustos
authors.forename: Mark A
authors.initials: MA
authors.lastname: Schumacher
authors.forename: Pietro D
authors.initials: PD
authors.lastname: Spanu
authors.forename: Robert
authors.initials: R
authors.lastname: Dickinson
authors.forename: Mervyn
authors.initials: M
authors.lastname: Maze
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Franks
mesh_terms: D000595:Amino Acid Sequence; D000777:Anesthetics / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D006801:Humans; D008195:Lymnaea; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry*; D024683:Potassium Channels, Tandem Pore Domain / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
chemical_list: D000777:Anesthetics; C498655:KCNK9 protein, human; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D011993:Recombinant Fusion Proteins; C420132:potassium channel subfamily K member 3