Molecular Bram Research, 14 (1992) 143-146 © 1992 Elsevier Science Pubhshers B V All rights reserved. 0169-328X/92/$05 00

143

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Cloning and functional expression of a cDNA encoding the mouse 132 subunit of the kainate-selective glutamate receptor channel Takao Morita a'c, Kenji Sakimura a, Etsuko Kushiya a, Masatoshi Yamazaki a, Hiroyuki Meguro a, Kazuaki A r a k i a, Teruo A b e b, Kazuhiro J. Mori c and Masayoshi Mishina a aDepartment of Neuropharmacology and bDepartment of Neurochemtstry, Brain Research lnstttute and 'Department of Btology, Faculty of Scwnce, Nugata Umverstty, Nugata (Japan) (Accepted 11 February 1992) Key words Glutamate receptor channel, Kamate receptor, cDNA cloning; cDNA expression, Xenopus oocyte

The primary structure of the mouse glutamate receptor f12 subumt has been deduced by cloning and sequencing cDNA The f12 subumt has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selectwe for kamate Glutamate receptor (GIuR) channels mediate fast excitatory synaptic transmission in the vertebrate central nervous system 13. Furthermore, Ca z+ permeation through glutamate-gated ion channels is suggested to play a key role in long-term modulation of synaptic efficacy and in neuronal cell death observed in acute and chronic brain disorders 3'4'19. Based on the action of selective agonists, three major classes of the GluR channel have been postulated: the a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, the kainate receptor and the N-methyl-o-aspartate (NMDA) receptor 17. A number of subunits of the GluR channel have been characterized at molecular level by cloning and expression of cDNAs 1"2'6'7"11"18'21"24. Structural analysis suggests that the GIuR subunits have four putative transmembrane segments characteristic for neurotransmittergated ion channels. The rat GluR1-GIuR4 subunits and the mouse a l (GIuR1) and a2 (GIuR2) subunits are highly homologous in amino acid sequence (about 70% identity) with each other 2"7AlAs'21, These homologous GluR subunits, classified as the a subfamily, form homomeric and heteromeric channels activated by L-glutamate, quisqualate, AMPA and kainate 2'7'11'1s'21. Quisqualate and A M P A show a higher apparent affinity for these GluR channels. Thus, the a subfamily represents the AMPA-selectxve GIuR channels. The rat GluR5 and GluR6 subunits have 80% amino acid sequence identity with each other and - 4 0 % identity with the members of the a subfamily (GIuR1-GluR4), thus representing an-

other subfamily L6. Although the functional property of the GluR5 subunit was not well characterized ~, the GIuR6 subunit has been shown to form kainate-selective GIuR channels 6. Furthermore, the rat KA-1 subunit showing - 4 0 % amino acid sequence identity with the GIuR1-GluR6 subunits exhibits hlgh-'h~finity kainate binding activities24. To isolate and characterize mouse GluR subunit cDNA clones, random-primed double-stranded cDNAs were prepared from ICR mouse forebrain poly(A) + RNA using cDNA synthesis system (Bethesda Research Labs.). D N A fragments longer than - 0 . 5 kilobase pairs (kb) were selected by electrophoresis on 1.5% agarose gel and were ligated with 2gtl0. The screening of the cDNA library was effected by hybridization at 37°C in the presence of 30% formamide with a mixture of the 0.6 kb BamHI fragment from pKCR30 (ref. 21) and the 0,96 kb HmcII fragment from pKCR24 (ref. 21) as probes. The hybridization solution contained 30% formamide, 0.9 M NaC1, 90 mM sodium citrate, 1% SDS, 100 pg/ml denatured salmon sperm DNA, and 0.1% each of Ficoll, polyvinylpyrrolidone and bovine serum albumin. The filters were washed in l x SSC (150 mM NaC1 and 15 mM sodium citrate) containing 0.1% SDS at room temperature and in 0.3x SSC containing 0.1% SDS at 42°C. Partial nucleotide sequence analysis 22 suggested that the clones S130, $121, YAM1 and YAM97 encoded the mouse counterparts of the GluR3, GIuR4, GluR5 and GluR6 subunits (designated as the a3, a4, fll and f12

Correspondence M Mlshlna, Department of Neuropharmacology, Brain Research Institute, Nngata Umverslty, 1-757 Asahlmachl, Nngata 951, Japan Fax: (81) 25-225-6458

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Fig 1 The deduced amino acid sequence of the mouse O l u R f12 s u b u m t and its ahgnment with those of the a l and ~z2 subumts N u m b e r s of the amino acid residues at the right-hand end of the md~vldual hnes are gp~en Sets of ~denucal amino acid residues m homologous region are enclosed The putanve t r a n s m e m b r a n e segments ( M 1 - M 4 ) are m&cated The asparagme reszdues as potential N-glycosylat~on s~tess and consensus phosphorylatlon sites for Ca2+-calmoduhn dependent protein kmase type II (refs 5, 20) m the predicted extracellular domain are marked with asterisks and open circles, respectwely The p o s m o n s of the point m u t a n o n s cd-K445E (ref 16) and a2-R586Q (refs 10, 16, 23) affecting the agomst binding and channel p r o p e m e s , respectp~ely, are indicated by arrowheads The amino acid differences between the mouse f12 subumt and the rat GluR6 s u b u m t 6 are marked with u n d e r h n e s Analys~s of nucleotlde and amino acid sequences was carried out using G E N E T Y X software (SDC) The nucleoude sequence of the f12 subumt wdl appear m D D B J , E M B L and Gene Bank Nucleonde Sequence Database under the accession n u m b e r D10054

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Fig 2. Funcnonal properties of the GIuR f12 subunit expressed in Xenopus oocytes. A' whole-cell currents act|rated by bath application of 100/~M L-glutamate (Glu), 10 pM kalnate (Kam) and 100 pM qmsqualate (Quis) were recorded from an oocyte rejected with the f12 subumt-speclfic mRNA (-20 ng) before and after treatment with 10 ~M Con A Membrane potential, -70 inV. Inward current is downward. The durat|on of agonIst apphcation is indicated by bars without taking into account the dead-space time in the perfuslon system (-2 s) B current (/)-voltage (V) relationship Wholecell currents activated by bath applicatmn of 10 pM kalnate were recorded under voltage clamp at different membrane potentmls from an oocyte injected with the f12 subumt-speclfic mRNA (-20 ng)

subunits), respectively. The primary structure of the mouse GluR/32 subunit was deduced from the nucleotide sequence of the clone YAM97 using a single large open reading frame (Fig. 1). The amino-terminal hydrophobic segment is assigned to represent a signal peptide as proposed for the GluR a l and a2 subunits 21. The expected mature ,62 subunit is composed of 833 amino acid residues with a calculated molecular weight of 93,891. The amino acid sequence of the mouse ,62 subunit is identical to that of the rat GIuR6 subunit 6 except for the residues 377 and 818-833. The mouse ,62 subunit shows 81% amino acid sequence identity with the rat GluR5 subunit 2. As shown m Fig. 1, an overall sequence homology is noted between the mouse ,62 subunit and the mouse a subunits (38-40% identity). Functional properties of the ,62 subunit were exam-

ined using a c D N A expression system. The 3.0 kb EcoRI fragment from clone YAM97 was blunted by the Klenow fragment and was inserted into the SmaI site of pSP64AX (ref. 25) in the same orientation as the SP6 promoter to yield the plasmid pSPGRfl2. The ,62 subunit-speclfiC m R N A was synthesized in vitro 12,15using EcoRI-cleaved pSPGR,62, as template. Xenopus oocytes were injected with the ,62 subunit-specific m R N A and whole-cell currents were recorded with a conventional two-micropipette voltage clamp 21. The mjected oocytes showed no detectable response (