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

2007; Elsevier BV; Volume: 282; Issue: 29 Linguagem: Inglês

10.1074/jbc.m610692200

1083-351X

Isabelle Andres-Enguix, Alex Caley, Raquel Yustos, Mark Schumacher, Pietro D. Spanu, Robert Dickinson, Mervyn Maze, Nicholas P. Franks,

Neuroscience and Neuropharmacology Research

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 IK(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 IK(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. 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 IK(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 IK(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. For more than 150 years, simple volatile organic molecules have been used as general anesthetics. How and where these agents act in the central nervous system to cause loss of consciousness and insensitivity to pain remains a mystery, although a great deal of progress has been made toward identifying plausible mechanisms and possible molecular targets (3Campagna J.A. Miller K.W. Forman S.A. N. Engl. J. Med. 2003; 348: 2110-2124Crossref PubMed Scopus (661) Google Scholar, 4Franks N.P. Br. J. Pharmacol. 2006; 147: S72-S81Crossref PubMed Scopus (311) Google Scholar, 5Rudolph U. Antkowiak B. Nat. Rev. Neurosci. 2004; 5: 709-720Crossref PubMed Scopus (639) Google Scholar, 6Sonner J.M. Antognini J.F. Dutton R.C. Flood P. Gray A.T. Harris R.A. Homanics G.E. Kendig J. Orser B. Raines D.E. Rampil I.J. Trudell J. Vissel B. Eger E.I. Anesth. Analg. 2003; 97 (2nd.): 718-740Crossref PubMed Scopus (285) Google Scholar). Although it is generally accepted that these drugs act by binding directly to protein targets, it is far from agreed what these targets are or whether the state of general anesthesia is due to effects at a relatively small number of critical molecular sites or due to the combined effects of small perturbations at a very large number of sites. The molecular targets that have most often been proposed to be responsible for the actions of volatile general anesthetics (3Campagna J.A. Miller K.W. Forman S.A. N. Engl. J. Med. 2003; 348: 2110-2124Crossref PubMed Scopus (661) Google Scholar, 4Franks N.P. Br. J. Pharmacol. 2006; 147: S72-S81Crossref PubMed Scopus (311) Google Scholar, 5Rudolph U. Antkowiak B. Nat. Rev. Neurosci. 2004; 5: 709-720Crossref PubMed Scopus (639) Google Scholar, 6Sonner J.M. Antognini J.F. Dutton R.C. Flood P. Gray A.T. Harris R.A. Homanics G.E. Kendig J. Orser B. Raines D.E. Rampil I.J. Trudell J. Vissel B. Eger E.I. Anesth. Analg. 2003; 97 (2nd.): 718-740Crossref PubMed Scopus (285) Google Scholar) are the γ-aminobutyric acid, type A receptor, the related glycine receptor, and certain members of a relatively recently discovered family of K+ channels, the two-pore domain K+ (2PK) 6The abbreviations used are: 2PK, two-pore domain; HEK-293, human embryonic kidney-293; TASK, two-pore domain acid-sensitive potassium; BLAST, basic local alignment search tool; RACE, rapid amplification of cDNA ends; TM, transmembrane. channels, so named because their primary sequences contain two pore-forming segments. The 2PK channels form a diverse and highly regulated superfamily of channels that are thought to provide baseline regulation of membrane excitability (7Goldstein S.A.N. Bockenhauer D. O'Kelly I. Zilberg N. Nature Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (570) Google Scholar, 8Lesage F. Lazdunski M. Am. J. Physiol. 2000; 279 (–F801): F793Crossref PubMed Google Scholar, 9Patel A.J. Honoré E. Trends Neurosci. 2001; 24: 339-346Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 10Talley E.M. Sirois J.E. Lei Q. Bayliss D.A. Neuroscientist. 2003; 9: 46-56Crossref PubMed Scopus (133) Google Scholar, 11Yost C.S. Curr. Drug Targets. 2003; 4: 347-351Crossref PubMed Scopus (34) Google Scholar, 12Honore E. Nat. Rev. Neurosci. 2007; 8: 251-261Crossref PubMed Scopus (391) Google Scholar). The possibility that neuronal excitability might be reduced by the activation of K+ channels has been long considered to be a plausible mechanism for general anesthesia (13Nicoll R.A. Madison D.V. Science. 1982; 217: 1055-1057Crossref PubMed Scopus (246) Google Scholar, 14Franks N.P. Lieb W.R. Nat. Neurosci. 1999; 2: 395-396Crossref PubMed Scopus (41) Google Scholar, 15Franks N.P. Honore E. Trends Pharmacol. Sci. 2004; 25: 601-608Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), and such an anesthetic-activated K+ current was first characterized in molluscan pacemaker neurons (1Franks N.P. Lieb W.R. Nature. 1988; 333: 662-664Crossref PubMed Scopus (168) Google Scholar, 16Lopes C.M.B. Franks N.P. Lieb W.R. Br. J. Pharmacol. 1998; 125: 309-318Crossref PubMed Scopus (35) Google Scholar). Subsequently, a family of 15 channels has been identified in mammals (7Goldstein S.A.N. Bockenhauer D. O'Kelly I. Zilberg N. Nature Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (570) Google Scholar, 8Lesage F. Lazdunski M. Am. J. Physiol. 2000; 279 (–F801): F793Crossref PubMed Google Scholar, 9Patel A.J. Honoré E. Trends Neurosci. 2001; 24: 339-346Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 10Talley E.M. Sirois J.E. Lei Q. Bayliss D.A. Neuroscientist. 2003; 9: 46-56Crossref PubMed Scopus (133) Google Scholar, 11Yost C.S. Curr. Drug Targets. 2003; 4: 347-351Crossref PubMed Scopus (34) Google Scholar, 12Honore E. Nat. Rev. Neurosci. 2007; 8: 251-261Crossref PubMed Scopus (391) Google Scholar) with several members being activated by volatile anesthetics. Most work has been done on TASK and TREK channels. Not only are these channels sensitive to anesthetics (17Patel A.J. Honoré E. Anesthesiology. 2001; 95: 1013-1021Crossref PubMed Scopus (93) Google Scholar), but recent work has also shown that genetically engineered animals that lack these channels have a diminished anesthetic sensitivity (18Linden A.M. Aller M.I. Leppa E. Vekovischeva O. Aitta-Aho T. Veale E.L. Mathie A. Rosenberg P. Wisden W. Korpi E.R. J. Pharmacol. Exp. Ther. 2006; 317: 615-626Crossref PubMed Scopus (75) Google Scholar, 19Heurteaux C. Guy N. Laigle C. Blondeau N. Duprat F. Mazzuca M. Lang-Lazdunski L. Widmann C. Zanzouri M. Romey G. Lazdunski M. EMBO J. 2004; 23: 2684-2695Crossref PubMed Scopus (443) Google Scholar). How anesthetics cause 2PK channel activation at the molecular level, however, is poorly understood. Using a combination of electrophysiology and molecular genetics, some key anesthetic determinants have been identified for both TASK and TREK channels (20Patel A.J. Honoré E. Lesage F. Fink M. Romey G. Lazdunski M. Nat. Neurosci. 1999; 2: 422-426Crossref PubMed Scopus (583) Google Scholar, 21Talley E.M. Bayliss D.A. J. Biol. Chem. 2002; 277: 17733-17742Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The identification of specific amino acids that are essential for anesthetic action provides a powerful tool for investigating the importance of the target in the effects of anesthetics in the whole animal. If an amino acid can be identified whose mutation affects only anesthetic sensitivity with little or no effect on other channel properties, then such a mutation can in principle be tested in whole animals to assess the importance of that channel for the in vivo anesthetic phenotype (22Jurd R. Arras M. Lambert S. Drexler B. Siegwart R. Crestani F. Zaugg M. Vogt K.E. Ledermann B. Antkowiak B. Rudolph U. FASEB J. 2003; 17: 250-252Crossref PubMed Scopus (513) Google Scholar, 23Reynolds D.S. Rosahl T.W. Cirone J. O'Meara G.F. Haythornthwaite A. Newman R.J. Myers J. Sur C. Howell O. Rutter A.R. Atack J. Macaulay A.J. Hadingham K.L. Hutson P.H. Belelli D. Lambert J.J. Dawson G.R. McKernan R. Whiting P.J. Wafford K.A. J. Neurosci. 2003; 23: 8608-8617Crossref PubMed Google Scholar). This approach requires two, ideally closely related, isoforms of the channel with very different anesthetic responses (usually differing sensitivities). One difference that has yet to be exploited in this regard is the fact that many general anesthetics show stereoselectivity in their actions (24Franks N.P. Lieb W.R. Nature. 1994; 367: 607-614Crossref PubMed Scopus (1652) Google Scholar). For the inhalational anesthetics this was first shown using isoflurane with the anesthetic-activated potassium current IK(An) in Lymnaea stagnalis (2Franks N.P. Lieb W.R. Science. 1991; 254: 427-430Crossref PubMed Scopus (247) Google Scholar). S(+)-Isoflurane was twice as effective at activating this current as the R(–)-enantiomer. We reasoned that knowledge of the amino acid sequence of the channel from Lymnaea might provide novel information that would help to identify the location of the anesthetic binding sites. In this report we describe the cloning of an anesthetic-activated channel from L. stagnalis and show how its unusual properties can be used to provide information that differentiates between the location of anesthetic binding sites and regions of the channel responsible for transducing anesthetic binding to changes in gating. We exploit the fact that hTASK channels differ greatly in the extent they are activated by the volatile agent chloroform. Using a combination of chimeric constructs (between hTASK-1 and hTASK-3 channels and hTASK-1 and LyTASK channels) and site-directed mutagenesis, we identify a specific amino acid that determines anesthetic sensitivity, and we use stereoselectivity as a criterion to determine its likely role as part of an anesthetic binding site. Isolation of Total RNA from the Central Nervous System of L. stagnalis—Adult L. stagnalis were obtained from Blades Biological, Ltd. (Edenbridge, Kent, UK) and kept in tanks of water at 22 °C until use. The circumesophageal ganglia from 3–4 animals were rapidly removed and placed into snail Ringers (see below). Total RNA was then extracted using TRIzol reagent (Invitrogen) together with Phase Lock Gel Heavy (Eppendorf, Cambridge, UK) to facilitate the separation between the organic and aqueous phases, and Pellet Paint CO-precipitant (Novagen, Nottingham, UK), a dye-labeled carrier, which allowed easy visualization and localization of the RNA pellet. DNA was removed by treatment with Amplification Grade DNase I (Invitrogen) (1 unit/μg of RNA). The DNase I was then removed by a phenol:chloroform:isoamyl alcohol extraction (25:24:1) followed by a chloroform:isoamyl alcohol (24:1) extraction (25Sambrook J. Russell D.W. Molecular Cloning. 2001; (, Third Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar). Degenerate Reverse Transcription-PCR—Total RNA from Lymnaea central nervous system (400 ng) was reverse transcribed with 50 ng of random hexamers in a 20-μl reaction volume using the SuperScript First-strand Synthesis System (Invitrogen). To exclude the contribution of genomic DNA to the final PCR step, incubations with and without reverse transcriptase were run simultaneously. The first strand cDNA was used as a template for PCR amplification using degenerate primers designed against stretches of conserved amino acid residues at the start and including part of the TM2 region (forward primer: 5′-CGS SGG AAR RGC WTT YTG YAT GTT CTA YG-3′; where S = C/G, R = A/G, W = A/T, and Y = C/T) and within the P2 region (reverse primer: 5′-CRW ART CRC CRA ARC CDA TWG TNG TYA R-3′; where D = A/G/T and N = A/C/G/T) of predicted and cloned 2PK channels. 2 μl of first strand cDNA was amplified (2 μm primers) using the HotStar-Taq Master Mix (Qiagen). PCR reaction mixtures were resolved on a 1.5% agarose gel and visualized with ethidium bromide. A distinct band of the expected size of ∼300 bp for the putative 2PK channel cDNA was observed. The PCR reaction mixture was purified using QIAquick columns (Qiagen), directly subcloned into the pCRII TOPO vector (Invitrogen) by TA cloning and sequenced on both strands using the universal M13 primers. Sequencing was done by MWG-Biotech (Ebersberg, Germany). Databases were searched for similar sequences using the BLAST alignment program (26Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (73420) Google Scholar). Rapid Amplification of cDNA Ends—5′-RACE and 3′-RACE PCR reactions were performed according to the manufacturer's instructions using the BD SMART RACE cDNA amplification kit (Clontech, St Germain en Laye, France) to obtain the complete 5′- and 3′-ends of the target LyTASK cDNA. The partial sequence of the LyTASK was used to design specific primers for 5′- and 3′-RACE. The primer used for the 5′-RACE was 5′-AGG AGA ATA TGG CCG CCC CCG ACG TGA G-3′. The primer used for the 3′-RACE was 5′-AGA GCG TGG GCG AGC GCC TCA AC-3′. Thermal cycling was performed using touchdown PCR. RACE products were isolated from a 1.2%-agarose gel using a Nucleotrap gel extract kit (Clontech), subcloned into pCRII TOPO vector and sequenced on both strands. The whole coding region of LyTASK cDNA was obtained by PCR using primers designed from the extreme 5′-end (5′-CGG AGA GCA CAC ATG CCT CCA-3′; sense) and 3′-end (5′-TAA ATC TAA TCA GCA ACT TGT GTT-3′; antisense) of LyTASK cDNA with the BD Advantage 2 PCR enzyme system (Clontech) and the 5′-RACE ready cDNA as template. Amplified products were subcloned into pCRII TOPO and sequenced on both strands. Sequence Analysis—L. stagnalis amplicons were used for translated blast searches (tBLASTx) of the NCBI non-redundant nucleotide sequence data base (www.ncbi.nih.gov/BLAST/). The TMpred program (27Hofmann K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 374: 166Google Scholar), which predicts membrane-spanning regions and their orientation using a data base of known transmembrane proteins, was used to predict the topology of the cloned LyTASK. Potential N-linked glycosylation sites were predicted using NetNGlyc 1.0. KinasePhos was used with a 95% prediction specificity to predict putative phosphorylation sites for protein kinases. ClustalW was used to align the deduced amino acid sequence of the cloned LyTASK with 2PK channels from other species. ALIGN was used with default settings to perform pairwise alignments of the deduced amino acid sequence of the cloned LyTASK with 2PK channels from other species. Phylogenetic trees were generated using ClustalW. Expression Vectors—For transfection into modified HEK-293 cells (tsA 201), 1.1-kb cDNA containing the entire coding region for LyTASK was subcloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen) via non-directional cloning by ligating into the EcoRI sites after cutting LyTASK/ pCRII TOPO (Invitrogen) with EcoRI. Expression vectors with LyTASK cDNA in the correct orientation were identified by restriction analysis with AgeI and XhoI. pcDNA 3.1(+) expression vectors with cDNA encoding human TASK-1 (GenBank™ accession number NM 002246) and human TASK-3 (GenBank™ accession number NM 016601) were a gift from Dr A. Mathie (Imperial College London). Site-directed Mutagenesis of TASK Channels—Site-directed mutagenesis was performed on TASK cDNA using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Complementary primers were designed so as to contain the desired mutation together with a silent mutation introducing a diagnostic restriction site. Mutant DNA constructs were verified by restriction digests and sequencing to confirm the introduction of the correct mutated bases (MWG-Biotech). Chimeric Constructs—Chimeras between the different TASK channels were generated by PCR using the overlap-extension method. The cDNAs encoding the chimeric channels were subcloned into pcDNA 3.1 and confirmed by sequencing. Cell Culture and Transfection into HEK-293 Cells—The HEK-293 cells were maintained under standard conditions in growth media (minimum essential media Eagle with Earle's salts, l-glutamine and sodium bicarbonate, supplemented with 1% non-essential amino acids, 1% penicillin (10,000 units/ml), streptomycin (10 mg/ml), and 10% heat-inactivated fetal bovine serum) in a 5% CO2:95% air, humidified incubator at 37 °C. For electrophysiological studies, cells were plated on glass coverslips previously coated with poly-d-lysine. Cells were transiently cotransfected with TASK/pcDNA3.1 and green fluorescent protein/pcDNA3.1 using the calcium phosphate precipitation method. Transfected cells were identified for electrophysiology using a Nikon microscope with an epifluorescence attachment. Electrophysiological recordings were performed within 24–48 h of transfection. Whole Cell Patch Clamp Recording from HEK-293 Cells—All chemicals and reagents were obtained from Sigma (Poole, Dorset, UK) or VWR International Ltd. (Lutterworth, Leicestershire, UK), unless otherwise stated. The composition of the normal control extracellular solution was (in mm) 140 NaCl, 2.5 KCl, 2 MgCl2, 10 HEPES, 10 d-glucose, 1 CaCl2, titrated to pH 7.4 with NaOH. Extracellular solutions containing elevated K+ were made by substituting NaCl with KCl. When the extracellular pH was a variable, the extracellular solution was titrated with either NaOH or HCl. For the experiments with zinc and copper, stock solutions of 1 mm ZnCl2 and 500 mm CuCl2, respectively, were prepared in extracellular solution and diluted to the desired test concentrations on the day of the experiment. For experiments with arachidonic acid a stock solution of 100 mm was made in ethanol, kept under nitrogen at –20 °C for no more than 3 days, and was diluted in extracellular solution to the test concentration (10 μm) before the experiments. In experiments with arachidonic acid, the control solution contained the same concentration of ethanol (1.7 mm) as the test solution. Patch electrodes were pulled from thickwalled borosilicate glass capillaries (GC150F-10, Harvard Apparatus, Edenbridge, Kent, UK) using a two-stage vertical puller (PP-830, Narishige, Tokyo, Japan), and their tips were fire-polished. Patch electrodes were back-filled with 0.2-μm-filtered intracellular solution, which consisted of (mm): 120 KCH3SO4, 4 NaCl, 1 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, 3 MgATP, 0.3 Na2-GTP, titrated to pH 7.20 with KOH. Electrode resistances ranged from 3 to 5 mΩ. Voltage clamp recordings were performed in the whole cell configuration using a patch clamp amplifier (Axopatch 200A, Axon Instruments, Union City, CA). Series resistance was compensated by 80% and monitored during the recording. Voltage commands were applied, and currents were recorded using pClamp 6 software (Axon Instruments). Cells were held at –80 mV and a linear voltage ramp from –120 mV to –0 mV in 2 s followed by a return ramp from 0 mV to –80 mV in 0.5 s, applied at 10-s intervals. Recordings with a positive holding current at –80 mV (indicating a negligible leak conductance) and an access resistance of <20 mΩ were included in the analysis. The output of the patch clamp amplifier was filtered at 100 Hz (–3 dB) using an 8-pole Bessel filter (Frequency Devices 902, Lyon Instruments, Ltd., UK) before being digitized at 500 Hz and recorded on a computer hard disk via an analog to digital converter (Digidata 1200, Axon Instruments). All experiments were performed at room temperature (22 ± 1 °C), and solutions were applied by a gravity-fed perfusion system. Currents were usually measured from the ramps at –50 mV where the contribution of endogenous currents from HEK-293 cells was negligible. Two-electrode Voltage Clamp Recording from L. stagnalis Neurons—Circumesophageal ganglia from L. stagnalis were excised, and the loose connective tissue overlying the right parietal ganglion was removed. This ganglion was then dissected away intact and held by its nerve root using light suction. The tough endoneurium was removed after softening in Pronase (∼2 mg/ml) for 10–15 min exposing the cluster of light yellow neurons used in these experiments (in the ventral part of the ganglion). The ganglion was held in a small chamber and continuously perfused with normal snail Ringers, which consisted of (in mm): 50 NaCl, 2.5 KCl, 4 CaCl2, 4 MgCl2, 10 HEPES, 5 glucose titrated to pH 7.4 with NaOH. Electrodes were pulled from 1-mm borosilicate glass capillaries and filled with 2.5 m KCl and had resistances between 10 and 30 mΩ. Following impalement, the chosen cell was carefully removed from the ganglion, and all experiments were performed on completely isolated neurons. A two-electrode voltage-clamp amplifier was used (Axoclamp 2A, Axon Instruments), with the current record being filtered (50 Hz, –3 dB) by an 8-pole Bessel filter. Data were digitized at 200 Hz and transferred to a computer for analysis. Steady-state I-V curves were constructed by slowly ramping the membrane potential (usually at 3 mV s–1) in the depolarizing direction. When the extracellular pH was a variable, the extracellular solution was titrated with either NaOH or HCl and the pH dependence of the anesthetic-activated potassium current IK(An) was measured from cells clamped at –40 mV. This minimized the small contribution of an anesthetic-activated inward current, which is present to varying degrees in Lymnaea right-parietal ganglion neurons and which reverses at ∼–40 mV (data not shown). Zinc- and copper-containing solutions were prepared as described above. Anesthetic Solutions—Anesthetic-containing solutions were made on the day of the experiment, and care was taken throughout to minimize losses of volatile anesthetics. Anesthetic solutions at a chosen concentration were prepared as volume fractions of a saturated aqueous solution. The concentration of the saturated solutions were taken to be 17.5 mm for halothane (28Raventós J. Br. J. Pharmacol. Chemother. 1956; 11: 394-410Crossref PubMed Scopus (116) Google Scholar), 66.6 mm for chloroform (29Firestone L.L. Miller J.C. Miller K.W. Roth S.H. Miller K.W. Molecular and Cellular Mechanisms of Anesthetics. 1986: 455-470Google Scholar), and 15.3 mm for isoflurane and its enantiomers (2Franks N.P. Lieb W.R. Science. 1991; 254: 427-430Crossref PubMed Scopus (247) Google Scholar). The optical isomers of isoflurane were prepared, and their purities were analyzed by Anaquest Inc. (Murray Hill, NJ). Chemical purities were 99.0% for S(+)-isoflurane and 99.1% for R(–)-isoflurane; the corresponding optical purity ratios were 99.5%/0.5% and 99.0%/1%, respectively. Statistics and Data Analysis—Data are expressed throughout as means ± S.E., unless otherwise stated. In the figures, when the error bars are not shown they are smaller than the size of the symbol. Concentration-response curves were usually fitted with Hill equations of the form (Equation 1), I=ImaxcnHcnH+EC50nH1 where I is the response, c the concentration, Imax the maximum response, EC50 the concentration for a 50% effect, and nH is the Hill coefficient. Any percentage change was calculated relative to the average of controls taken just before and just after a test application. Statistical significance was assessed using the Student's t test. Molecular Cloning and Sequence of LyTASK—Degenerate oligonucleotides were designed to hybridize in PCR reactions with conserved regions of 2PK channel cDNAs derived from total RNA extracted from L. stagnalis CNS. Comparison with the databases using TBLASTX identified a cDNA fragment for which the derived amino acid sequence was similar to the region extending from the start of the second transmembrane domain to the end of the second pore domain of previously cloned mammalian 2PK TASK channels. The whole coding region for the channel was obtained using 5′- and 3′-RACE PCR, followed by reverse transcription-PCR using specific primers based on the predicted N and C termini. The cDNA sequence contained an open reading frame of 1083 bases that encoded a 361-amino acid polypeptide with a calculated molecular mass of 41 kDa (Fig. 1A). Four transmembrane segments (TM1 to TM4) were predicted (see Fig. 1B) using the TMpred program (27Hofmann K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 374: 166Google Scholar). Two poreforming regions (P1 and P2) containing the TXG(Y/I/F)G consensus motif of potassium channels (see Fig. 1A) also scored highly using this algorithm. The putative K+ channel subunit exhibits a short intracellular N-terminal domain, a large extracellular loop between TM1 and P1, and a large intracellular carboxyl domain (see Fig. 1B), consistent with other known 2PK channels (7Goldstein S.A.N. Bockenhauer D. O'Kelly I. Zilberg N. Nature Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (570) Google Scholar, 8Lesage F. Lazdunski M. Am. J. Physiol. 2000; 279 (–F801): F793Crossref PubMed Google Scholar, 9Patel A.J. Honoré E. Trends Neurosci. 2001; 24: 339-346Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). The Lymnaea subunit may potentially undergo several post-translational modifications (see Fig. 1A). One putative N-linked glycosylation site is present in the TM1-P1 loop (Asn-53). Putative intracellular phosphorylation sites for several protein kinases were identified, including two for protein kinase A at Ser-10 and Thr-152; one for protein kinase C at Ser-338; and two for casein kinase II at Thr-334 and Ser-340. An alignment of the amino acid sequence of the Lymnaea subunit with sequences of mammalian 2PK channel members of the TASK subfamily is shown in Fig. 2. The Lymnaea subunit shares ∼47% overall amino acid identity with human and rat TASK-1 and TASK-3 and ∼41% with human and rat TASK-5. The Lymnaea subunit (which we refer to as LyTASK) has a lower degree of similarity to the other cloned mammalian 2PK channels with 20–27% amino acid identity. Sequence identity of the Lymnaea subunit to TASK-1, TASK-3, and TASK-5 is much higher in the core region comprising the transmembrane domains, the extracellular loop, and the pore regions (60% amino acid identity with hTASK-1 and hTASK-3) rather than in the C-terminal domain (24% amino acid identity with hTASK-1, 30% with hTASK-3). The alignment (Fig. 2A) reveals a number of features that are common to TASK-1, TASK-3, TASK-5, and the Lymnaea subunit. It also highlights several features that are not shared between them. The two amino acid motifs P1 and P2 that form the selectivity filter of a 2PK channel are conserved in LyTASK, TASK-1, TASK-3, and TASK-5. LyTASK N-linked glycosylation site (Asn-53) is conserved in the mammalian TASK-1, TASK-3, but not in TASK-5 (see Fig. 2A). TASK-1, TASK-3, TASK-5, and LyTASK all contain a histidine residue immediately downstream of the "GYG" pore motif in the P1 domain (His-98). The protonation of this His-98 has been shown to be critical for the pH sensitivity of TASK-1 (30Morton M.J. O'Connell A.D. Sivaprasadarao A. Hunter M. Pflugers Arch. 2003; 445: 577-583Crossref PubMed Scopus (58) Google Scholar, 31Yuill K. Ashmole I. Stanfield P.R. Pflugers Arch. 2004; 448: 63-69Crossref PubMed Scopus (15) Google Scholar) and TASK-3 (32Rajan S. Wischmeyer E. Xin Liu G. Preisig-Muller R. Daut J. Karschin A. Derst C. J. Biol. Chem. 2000; 275: 16650-16657Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). TASK-1, TASK-3, TASK-5, and LyTASK also contain an aspartate residue (Asp-204) in the equivalent position in the P2 domain, which has been shown to be important for both normal pH sensitivity and K+ selectivity of TASK-1 (31Yuill K. Ashmole I. Stanfield P.R. Pflugers Arch. 2004; 448: 63-69Crossref PubMed Scopus (15) Google Scholar). This would suggest that the Lymnaea protein is an acid-sensing K+ channel (see below). In contrast to mammalian TASK-3, which contains a glutamic acid at position 70, which confers sensitivity to zi