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J. Biol. Chem., Vol. 282, Issue 29, 20977-20990, July 20, 2007

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Determinants of the Anesthetic Sensitivity of Two-pore Domain Acid-sensitive Potassium Channels

MOLECULAR CLONING OF AN ANESTHETIC-ACTIVATED POTASSIUM CHANNEL FROM LYMNAEA STAGNALIS^*

Isabelle Andres-Enguix¹², Alex Caley¹³, Raquel Yustos, Mark A. Schumacher⁴, Pietro D. Spanu, Robert Dickinson^¶, Mervyn Maze^¶, and Nicholas P. Franks^¶5

From the Biophysics Section, Blackett Laboratory, and Division of Biology, Imperial College, South Kensington, London SW7 2AZ and the ^¶Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College School of Medicine, Chelsea and Westminster Hospital, London SW10 9NH, United Kingdom

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 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.

Received for publication, November 17, 2006 , and in revised form, April 13, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) EF640973 [GenBank] .

¹ Both authors contributed equally to this work.

² Recipient of a studentship from the Westminster Medical School Research Trust.

³ Recipient of a Biotechnology and Biological Sciences and Research Council CASE studentship with Air Products and Chemicals Inc. Present address: School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.

⁴ Present address: Dept. of Anesthesia and Perioperative Care, 513 Parnassus Ave., University of California San Francisco, San Francisco, CA 94143-0427.

⁵ To whom correspondence should be addressed: Biophysics Section, Blackett Laboratory, Imperial College, South Kensington, London SW7 2AZ, United Kingdom. Tel.: 44-20-7594-7629; Fax: 44-20-7589-0191; E-mail: n.franks{at}imperial.ac.uk.

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