1722 Anesthesiology 2000; 92:1722-30 0 2000 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Volatile Anesthetics Activate the Human Tandem Pore Domain Baseline K+ Channel KCNK5 Andrew T. Gray, M.D., Ph.D.,* Byron 6. Zhao, Ph. D.,t Christoph H. Kindler, M. D.,* Bruce D. Winegar, Ph. D.,§ Matthew J. Mazurek, B.A.,II Jie Xu, B.A.,t Raymond A. Chavez, Ph.D.,t John R. Forsayeth, Ph.D.,# C. Spencer Yost, M.D. ** Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/6/1722/430285/0000542-200006000-00032.pdf by guest on 01 October 2021 Background. Previous studies have identified a volatile anes- by protein kinase C. Clinical concentrations of volatile general thetic-induced increase in baseline potassium permeability and anesthetics potentiated KCNK5 currents by 8-30°/o. concomitant neuronal inhibition. The emerging family of tan- Conclusion: Human KCNK5 is a tandem pore domain potas- dem pore domain potassium channels seems to function as sium channel exhibiting delayed activation and sensitivity to baseline potassium channels in vivo. Therefore, we studied the volatile anesthetics and may therefore have a role in suppress- effects of clinically used volatile anesthetics on a recently de- ing cellular excitability during general anesthesia. (Key words: scribed member of this family. Background channel; leak current; mammalian; outward recti- Methods: A cDNA clone containing the coding sequence of fier; pH.) KCNK5 was isolated from a human brain library. Expression of KCNK5 in the central nervous system was determined by North- POTASSIUM (K+) channels have important functions in ern blot analysis and reverse-transcription polymerase chain many tissues. In nonexcitable tissues, Kt channels mod- reaction. Functional expression of the channel was achieved by ulate the resting membrane potential, K+ homeostasis, injection of cRNA into Xenopus laevis oocytes. Results: Expression of KCNK5 was detected in cerebral cor- and signal transduction. In excitable tissues, K+ chan- tex, medulla, and spinal cord. When heterologously expressed nels also control action potential initiation, frequency, in Xenopus oocytes, KCNK5 currents exhibited delayed activa- and duration. At this time the largest mammalian families tion, outward rectification, proton sensitivity, and modulation of cloned K+ channels are voltage-gated channels (Kv series) and inward rectifiers (Kir series). These channels * Assistant Professor, Department of Anesthesia and Perioperative contain one pore-forming domain within their primary Care, University of CaIifornia, San Francisco, CaIifornia. amino acid sequence and form functional ion channels t Research Scientist, Elan Pharmaceuticals, Menlo Park, California. as tetrameric complexes. $ Visiting Assistant Professor, Department of Anesthesia and Periop- Recently, Kt channel subunits with two pore domains erative Care, IJniversity of California, San Francisco, California. in tandem have been cloned.' This structural feature Assistant Adjunct Professor, Department of Anesthesia and Periop- may allow ion channels to form as homodimers with erative Care, University of California, San Francisco, California. four pore domains lining the ion conduction pathway.' 11 Medical Student, Department of Anesthesia and Perioperative Care, The basic structure of mammalian tandem pore domain University of California, San Francisco, California. Kt channels includes four membrane-spanning domains # Principal Scientist, Elan Pharmaceuticals, Menlo Park, California. and a relatively large extracellular segment (50 -70 ** Associate Professor, Department of Anesthesia and Perioperative amino acids) preceding the first pore region.' Although Care, University of California, San Francisco, California. the genome of Caenorhabditis elegans contains a large Received from the Department of Anesthesia and Perioperative Care, number (2 40) of tandem pore domain Kf channel University of California, San Fran 0,California; and Elan Pharmaceu- subunits,' only seven mammalian subunits of this type ticals, Menlo Park, California. Submitted for publication May 12, 1999. Accepted for publication February 3, 2000. Supported by grants no. have been reported. GM57529 (to Dr. Winegar), GM57740 (to Dr. Forsayeth), and The functional properties of mammalian tandem pore GM51372 and GM58149 (to Dr. Yost) from the National Institutes of domain Kt channels are diver~e.~The family includes Health, Bethesda, Maryland. Presented in part at the annual meeting of the weak inward rectifiers IWIK-1 and TWIK-2, the the American Society of Anesthesiologists, Dallas, Texas, October 1 1, outward rectifier TREK-1, and the open rectifiers TASK-1 1999. and TRAAK (fig. 1A). Open rectification implies that Address reprint requests to Dr. Yost: Department of Anesthesia and currents satisfy the Goldman-Hodgkin-Katzcurrent equa- Perioperative Care, 513 Parnassus Avenue, Room S-261, Box 0542, San Francisco, California 94 143. Address electronic mail to: tion for an open channel. Although TWIK-l, TWIK-2, [email protected]~1 TREK-1 and TASK-1 are expressed in many tissues, Anesthesiology, V 92, No 6, Jun 2000 1723 A MAMMALIAN VOLATILE ANESTHETIC-ACTIVATED Kt CHANNEL B KCNKl (TWIK-1) c-, - I/n AA KCNK3 (TASK-1) - KCNK2 (TREK-1) Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/6/1722/430285/0000542-200006000-00032.pdf by guest on 01 October 2021 - N' N' I KCNK4 (TRAAK) - SC KCNKB (TASK-2) Fig. 1. (A) Predicted membrane topology of KCNK5 and other mammalian tandem pore domain K+ channels. Outer (0) and inner (i) sides of the plasma membrane are indicated. (B) Phylogenetic tree of the known members of the mammalian tandem pore domain Kf channel family. TRAAK is exclusively expressed in the central nervous this K+ channel and its basic physiology were previously system.' Tandem pore domain K+ channels are not described.2' This channel has been named KCNK5 be- gated by physiologic ligands or voltage and therefore cause it is the fifth member of the mammalian tandem serve as baseline (or background) channels.' pore domain K+ channel family isolated (potassium Physiologic baseline Kt channels have been described channel subfamily K). in a number of mammalian tissues, including pancreas,' corneal endothelia,' sympathetic ganglia, '"carotid bod- ies,' ' and thin myelinated axons. l2 Electrophysiologic properties of cloned outward and open rectifier baseline Methods K+ channels closely resemble baseline K+ currents that underlie the resting membrane potential of vertebrate Isolation of a cDNA Clone myelinated Thus, these channels likely con- A 360 -base pair expressed sequence tag from human tribute to the resting membrane conductance of myelin- prostate epithelium (accession AA533 124) was identi- ated nerves that limits action potential duration. fied with homology to the second pore domain of mam- Baseline K+ channels also may have a role in mediating malian tandem pore potassium channels by tBLASTn effects of volatile anesthetics. l4 The concept that volatile query.24 These BLAST (Basic Local Alignment Search anesthetics enhance neuronal Kt permeability has re- Tool) queries consisted of protein sequences of previ- ceived increasing experimental support over the past 20 ously cloned mammalian tandem pore domain potas- yr. Hyperpolarization induced by volatile anesthetics has sium channels used to identlfy homologous nucleotide now been described in a wide variety of cortical, hip- sequences of expressed sequence tags. To obtain full- pocampal, and spinal Most recently, Sirois neuron^.'^-'^ length clones, we screened a human lung cDNA library et al. l9 found that hypoglossal motor neurons display an from Gibco BRL (Gaithersburg, MD) and a human brain increase in K+ conductance with application of clinical cDNA library from Edge Biosystems (Gaithersburg, MD) concentrations of volatile anesthetics. These results in- with modified solution hybridization technique^.^^ One dicate that volatile anesthetics can increase K+ conduc- tance in neuronal tissue, leading to hyperpolarization full-length clone was found from the BRL library, and and decreased responsiveness to neuronal excitation. multiple clones were found from the Edge library. Both Specific volatile anesthetic-activated Kt channels were strands of one clone from the Edge library were se- first identified in invertebrate nervous Re- quenced and determined to be full length. This clone cently, Pate1 et al.** showed that baseline currents was therefore chosen for expression studies, and its passed by the mammalian tandem pore domain K+ chan- insert was subcloned into the POX vector to enhance iiels TREK-1 and TASK-1, when expressed in cultured efficiency of expression in oocytes.*6Sequence analysis cells, can be activated by volatile anesthetics. and alignments were performed with LaserGene (DNAS- Here we report the functional expression, tissue dis- TAR Inc., Madison, WI) and the ExPASy server (Uni- tribution, and anesthetic activation of another mamma- versity of Geneva, Geneva, Switzerland) to search the lian tandem pore domain K+ channel. The cloning of Prosite database. Anesthesiology, V 92, No 6,Jun 2000 1724 GRAY ET AL. Northern Blot Analysis und Reverse-Transcription periments were performed using frog Ringer's solution Polymerase Chain Reaction (composition: 115 rnM NaCl, 2.5 mM KC1, 1.8 mM CaCl,, A 500 -base pair restriction fragment was excised from 10 mM HEPES, pH 7.6) or high K+ frog Ringer's solution the expressed sequence tag clone (AA533124) by EcoRI (composition: 2.5