Molecular Cloning and Functional Expression of cDNA Encoding a ...

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THE JOURNAL OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 37, Issue of September 16, pp. 23274-23279, 1994 Printed in U.S.A.

Molecular Cloning and Functional Expressionof cDNA Encoding a Second Class of Inward Rectifier Potassium Channels in the Mouse Brain* (Received for publication, January 19, 1994, and inrevised form, June 14, 1994)

Naohiko TakahashiS, Ken-Ichirou MorishigeS, Arshad JahangirS, Mitsuhiko YamadaS, Ian Findlayl, Hidekazu KoyamaS, and YoshihisaKurachiSflll** From the $Llivision of Cardiovascular Diseases, Department of Internal Medicine, Wepartment of Pharmacology, Mayo Clinic, Rochester Minnesota 55905, SCNRS EP 21, Universite de Tours, Tours 37200, France, a n d the [Department of Pharmacology 11,Faculty of Medicine, Osaka University, Suita, Osaka 565, J a p a n

We have cloned a second class of inward rectifier po- sical voltage-gated potassiumchannels (7), since they probably tassium channels, designated MB-IRK2, from a mouse possess only twotransmembrane segments with one pore-formbrain cDNA library. The amino acid sequence of this ing region. Northern blot analysis demonstrated that t h e IRKl IRK1. Xenopus mRNA is expressed in the central nervous system (3). We acclone shares70%identity with the mouse oocytes injected withcRNA derived from MB-IRK2 ex- tually cloned a eDNA identical to mouse macrophage IRKl pressed a K+ current, which showed inward rectifying from a mouse brain cDNA library (MB-IRKl)@). channel characteristics similar to MB-IRK1 the current. In the present study, we have isolated and characterized the In contrast to the MB-IRK1 current, however, the MB- second member of the IRK family from a mouse brain cDNA IRK2 current exhibited significant inactivation during library, MB-IRK2. The single channel conductance, the kinetic hyperpolarizingpulses.Inpatchclampexperiments properties and t h e tissue distribution of MB-IRK2 were distinct with 140 l l l ~K+in the pipette, the single channel confrom thoseof MB-IRK1. The resultssuggest that MB-IRK1 and ductance of MB-IRH2 was 34.2 f 2.1 picosiemens (n= 5), MB-IRK2 are expressed differentiallyand may play similar but a value significantly larger than of that MB-IRKl(22.2 3.0 picosiemens, n = 5). Consistent with the whole cell distinct functional roles in the brain. current, the steady-state open probability (Po) ofthe EXPERIMENTALPROCEDURES MB-IRK2 channeldecreasedwithhyperpolarization, of Mouse Brain cDNA Library and DNA Sequencing-A Screening whereas thatof the MB-IRK1 remainedconstant. Northmouse brain cDNA library (Stratagene, La Jolla, CA) was screened ern blot analysis revealed the mRNA for MB-IRK2to be under a low stringency condition using a BstXI digested MB-IRKl(- 1.7 expressed in forebrain, cerebellum, heart, kidney, and kb)’ as a probe (8). 2 x lo6 phage cloneswere screened with a skeletal muscle. In the brain, the abundance of mRNA [~~-~~P]CTP-labeled probe. Hybridization was conductedin 5 x SSC (the for MB-IRK!Z was much higher in cerebellum than in composition of SSC was 0.15 M NaCl with 0.015 M sodium citrate, pH forebrain and vice uerm in the case of MB-IRK1. These 7.0), 30% formamide, 0.08%bovine serum albumin, 0.08% Ficoll, 0.08% 0.25 M Na,HPO,, 250 pg/ml denatured results demonstrate that theIRK family is composed of polyvinylpyrrolidone, 0.1% SDS, salmon sperm DNA, at 37 “C for 17 h. Filters were washed with 2 x multiple genes, which may play heterogenous functional SSC, 0.1% SDS at room temperature for 20 min and then exposed to roles in various organs, including the central nervous x-ray film overnight at -70 “C with an intensifymg screen. DNA sesystem. quencing was performed on both strands using a sequencing kit (U. S. BiochemicalCorp.) byspecific oligonucleotide primers as described previously (9). Functional Expression of a Mouse Brain InwardRectifier Potassium Inward rectifier potassium channels play a significant rolein the maintenance of t h e resting membrane potential, in regu- Channel in Xenopus Oocytes-The obtained positive clones were tranlating the duration of the action potential and in controlling scribed in vitro byT, RNA polymerase after digestion with appropriate restriction enzymes (8).These transcripts were dissolvedin sterile waexcitability of a variety of cell types including central nervous ter and injected to manually defoliculated oocytes (50 nl of 500 ng/pl). system cells (I, 2 ) . After injection, oocytes wereincubated in a modified Barth solution at An inward rectifier potassium channel (IRK11 has been re- 18 “C, and electrophysiologicalstudies were undertaken 48-96 h later. cently cloned froma mouse macrophage cell line (3) and shows Complementary RNA encoding MB-IRK1was also injected to defolicuconsiderable homology with an ATP-regulated potassium chan- lated oocytes (50nl of 500 ng/pl).Experiments were performedat room nel cloned from the outer medulla of rat kidney (ROMK1) (41, temperature (20-22 “C). Two-electrode voltage clamp experiments were carried out with a and a G-protein regulated potassium channel cloned from rat commercially available amplifier (Turbo Clamp TEC OlC, Tamm,Gerheart (GIRK1 or KGA) (5, 6). These channels differ from clas- many) using microelectrodes which, when filled with 3 M KCl, had resistances of 0.5-1.5 megaohms. Oocytes were bathed in a solution * This work was supported in part by a National Institutes of Health which contained 90 m KC1,3 mM MgCl,, 5 l l l ~HEPES, pH 7.4,and 300 Grant R 0 1 HL47360 (to Y. Kurachi). The costs of publication of this )ul niflumic acid to block endogenous chloride current. Oocytes were article were defrayed in part by the payment of page charges. This voltage-clamped at various holding potentials and voltage steps of 1.5-s article must therefore be hereby marked “advertisement”in accordance duration were applied to the cells in 10-mV increments every 5 6 . Single channel recordings were performedin the cell-attached patch with 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequencefs) reported in this paper has been submitted configuration using a patch clamp amplifier (Axopatch 200A Axon Into the GenBankm/EMBL Data Bank with accessionnumber(s) X80417. struments, Foster City, CA or EPC-7, List Electronic, Darmstadt, Ger** This work has been performedat the Mayo Clinic (Rochester,M N ) many). Both pipette and bathing solutions contained 140 mM KCI, 1.4 during the tenure of the established investigatorship of the American mM MgCl,, and 10 mM HEPES, pH 7.4. Heart Association (to Y. Kurachi). To whom correspondence should be Electrophysiological data were stored on video tapes using a PCM addressed: Dept. of Pharmacology 11, Faculty of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Tel.: 81-6-879-3510; * The abbreviations used are: kb, kilobase pair(s); S, siemens. Fax: 81-6-879-3519.

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FIG.1. Nucleotideand deduced amino acid sequence of MB-IRK2. Nucleotides are numbered in the 5'-3' direction, beginning with the first residue of ATG triplet encoding the methionine initiation site. The number of the nucleotide residue at theright end of each line is given. The deduced amino acidsequence is shown below the nucleotide sequence.Amino acid residues are numbered beginning with the methionine initiation site. Numbers of the last residue are given on the right-hand site. The non-sense codon TGA at the end of open reading frame is marked by dots. Proposed transmembrane segments M1, M2, and pore-forming domain H5 are underlined.

data recording system (VR-lOB, Instrutech Corp., New York). Foranalysis, the data were reproduced, low pass-filtered at 600 Hz (-3 dB) by an 8-pole Bessel filter (Frequency devices, Haverhill, MA), sampled at 3 kHz and analyzed off-line ona computer (MacintoshQuadra 700, Apple Computer Inc., Cupertino, CA) with a standard program (EP Analysis, Human Intelligence Inc., Rochester, MN). In the single-channel analysis, the threshold t o judge the open state was set at half of the unit amplitude of the single channel current. Statistical data areexpressed as mean 2 S.D. Northern Blot Analysis-Pieces of various tissues (forebrain, cerebellum, heart, kidney, and leg skeletal muscle)were isolated from 12-weekold female BALB/c mice. Total RNAs were extracted by the guanidium thiocyanate method (101,and poly(A)+ RNAs wereisolated using Oligotex-dTmRNAkit (QIAGEN, Chatsworth, CAI. Aliquots of 3 pg of poly(A)+ RNA were separated by electrophoresis in 1.0%agarose gel and blotted onto a Hybond-N nylon membrane (Amersham Corp.). Hybridization was conducted in 5 x SSC, 50%formamide, 0.08% Ficoll, 0.08% polyvinylpyrrolidone, 0.1% SDS, 0.25%Na2HPO,,250 pg/mldenatured salmon sperm DNA, at 42"C for 17 h. Glyceraldehyde-3-phosphatedehydrogenase cDNA probe was used as a control to ascertain that equivalent amounts of mRNAhad been transferred. Blots werewashed a t 60 "Cfor 15 min in 0.05 x SSC containing 0.1% SDS. The filter was exposed to Kodak XAR 5 film at -70 "C for 2 days with an intensifylng screen.

RESULTS

Molecular Cloning of an Inward Rectifier Potassium Channel-After screening of 2 x lo5 mouse brain cDNA clones, we obtained six positive clones. These phage inserts were subcloned into plasmids by rescue excision. The partial sequence analyses indicated that five of these six clones were full or partial clones encoding MB-IRK1, whereas the remainingone (MB26) had thenucleotide sequence nearly identical to that of RB-IRK2 (11).Accordingly, we designated thisclone MB-IRK2. The size of the MB-IRK2 clone obtained was 1777 base pairs. The nucleotide sequencing of MB-IRK2 revealed an open reading frameof 1281 base pairs, encoding a protein of 427 amino acids (Fig. 1).The deduced amino acid sequence of MB-IRK2 showed 97 and 70% identity with RB-IRK2 and MB-IRK1, respectively. The Kyte-Doolittle hydropathy plot (12) indicates two membrane-spanning hydrophobic segments (M1 and M2) with one pore-forming region (H5). Electrophysiological Characteristics of MB-IRK2 CurrentFig. 2 illustrates the results obtained from Xenopus oocytes which had been injectedwith cRNA, derived from MB-IRK2,96

Rectifier K+ Channels

Second Class of Brain Inward

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FIG.2. Cell currents recorded from Xenopus oocytes expressing theMBIRK2 clone. A, effect of varying the external K+ concentration. The holding potential was set a t the zero current level in each solution, i.e. at 0 mV in 90 mM K', at -17.4 mV in 45 mM K', a t -37.9 mV in 20 II~MK', at -55.3 mV in 10 m M K', and at -78.4 mV in 4 mM K'; the values correspond to the equilibrium potential for K+ at each concentration of external K+,with an assumption that the intracellular K+ concentration of oocytes is 90 mM (24). Traces elicited by steps from each holding potential to +50, +20, -10, -40, -70, -100, -130, and -160 mV are shown. B , currentvoltage relationships in solutions of 90 mM (O), 45 m (O),20 mM (V), 10 mM (A), and 4 mM (0) K'. K+ was substituted with Na'. Current amplitudes 10 ms from the start of voltage pulses are plotted. C and E illustrate currents induced by voltage steps from 0 mV to, in descending order, +50, +20, -10, -40, -70, -100, -130, and -160 mV. C , the effect of external Ba2+.D, current-voltage relationships of the steady-state currents recorded from this oocyte in solutions of 0 m (015, (O), and 50 Ba2+(V). E,the effects of external Cs'. F, current-voltage relationships of the steady-state currents recorded in solutions of 0 PM (01, 5 PM (01, and 50 p Cs+(V).

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FIG.3. Singlechannelrecordings from cell-attachedmembrane patches of Xenopus oocytes expressing the MB-IRK1 or MB-IRK!Z channel. A, membrane current traces recorded at the membrane potential values indicated to the left of each trace. The arrows to the right of certain traces indicate the patch current level recorded when all channels wereclosed. Each of these patches appeared to contain one MB-IRK1 or MBIRK2 inwardly rectifier potassium channel. B , the current-voltage relationships of the channel records shownin A.

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h before the recording. Under control condition with 90 nm of extracellular K+ ([K'],) (top truces in Fig. 2, A, C, and E ) , hyperpolarizing voltage steps from a holding potential of 0 mV revealed rapid activation (