Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1443-1447, February 1992 Neurobiology

Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors (neurotoidcity/human genome/ionic channels/membrane receptors/N-methyl-D-aspartate receptors)

WILLIAM SUN*, ANTONIO V. FERRER-MONTIEL*, ALEJANDRO F. SCHINDER*, JOHN P. MCPHERSONt, GLEN A. EVANS4:, AND MAURICIO MONTAL*§ *Departments of Biology and Physics, University of California, San Diego, La Jolla, CA 92093; tDepartment of Biological Chemistry, University of California, Irvine, CA 92717; and *Molecular Genetics Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037 Communicated by Morris E. Friedkin, October 30, 1991 (received for review September 6, 1991)

forts toward the isolation of clones from human brain and the functional characterization of the gene products. Localization of cloned glutamate receptors to human chromosomes would suggest plausible molecular explanations for human diseases that have been mapped and would assist in developing molecular markers for clinical screening programs (12). Availability of human brain receptors would allow in vitro testing of drugs that enhance or block receptor functions and would offer the potential to refine the design of central nervous system-specific glutamate receptor modulators that may prove of therapeutic value in neurology and psychiatry. We report here a step toward the realization of these goals: the isolation from human brain of two glutamate receptor cDNAs, HBGR11 and HBGR2, their mapping to human chromosome 5q31.3-33.3 and chromosome 4q25-34.3, respectively, and a pharmacological characterization of the channel encoded by the HBGR1 clone after expression in Xenopus oocytes.

A full-length cDNA clone encoding a glutaABSTRACT mate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCR analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists with the following specificity sequence: domoate > kainate >> quisqualate 2 ar-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid 2 L-glutamate >> N-methyl-D-aspartate. The kainateelicited currents were specifically blocked by 6-cyano-7nitroquinoxaline-2,3-dione but were insensitive to 2-amino-5phosphonovalerate and kynurenic acid. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

MATERIALS AND METHODS cDNA Library, PCR, and Hybridization Screening. A cDNA library was constructed in collaboration with Stratagene (13) by using mRNA isolated from a surgical section of human frontal cortex. The library was split into two fractions, one consisting of inserts 2-4 kilobases (kb) in length and the other enriched for inserts >4 kb. cDNAs were cloned into the A Zap II vector at the EcoRI site. DNA from an aliquot of the amplified titer was used as template for the PCR (14, 15). PCR primers were selected from sequences of rat brain glutamate receptors (7, 8) to encompass segments of low nucleotide degeneracy and high amino acid sequence conservation. Degenerate PCR primers were as follows: (i) GCGAATTCIGTSRRRGAYGGNAARTAYGG (where S = GorC, R = GorA, Y = TorC, and N = A, C, G, orT), sense primer coding for the sequence V(GKS)DGKYG (amino acids 441-447 of clone GluRl); (ii) GCGGTA-

Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system. Glutamate receptors are known to be involved in long-term potentiation, learning, and memory (1). Central nervous system glutamate receptor dysfunction has been implicated in conditions involving injury and neurotoxicity and in pathological states, such as epilepsy, Parkinson, Huntington, and Alzheimer diseases (2-4). Pharmacological and electrophysiological studies indicate that neurons express a heterogeneous population of receptor subtypes. Four major classes of receptors, all cation-selective channels, are well characterized: N-methyl-Daspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and 2-amino-4phosphonobutanoate (4, 5). Recently, a rat NMDA receptor clone was reported (32). The kainate and AMPA receptor subtypes have been cloned from the mammalian brain (6-8); yet, it is still unclear whether AMPA and kainate receptors are actually two distinct classes of receptors (9). In addition, a metabotropic receptor coupled to a G protein was isolated (10, 11). The central involvement of glutamate receptors in human central nervous system pathology has prompted ef-

CCITCRTACCACCAYTTRTTYTT, antisense primer cod-

ing for the sequence KNKWWYD (amino acids 759-765 of clone GluRl). The 5' end of the sense primer has an EcoRI site, whereas that of the antisense primer has a Kpn I site, indicated by a bar (j) separating these from the coding sequences. The PCR amplified a 1-kb fragment that was subcloned into the pBluescript KS(+) vector (Stratagene). Sequencing identified the 1-kb fragment as part of a gene highly homologous to rat brain clone GluRl or GluRA (7, 8), and the fragment was radiolabeled for hybridization screening of the human brain cDNA library. An in vivo excision Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; I, current; NMDA, N-methyl-D-aspartate; V, applied voltage. Standard one-letter amino acid code is used. §To whom reprint requests should be addressed. IThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M81886).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1443

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procedure (Stratagene) yielded the purified clones in the pBluescript SK(-) vector. Nucleotide Sequence. DNA sequencing was according to Sanger et al. (16) with the use of a Sequenase kit (United States Biochemical). Open reading frames were compared for sequence homology with published sequences by using the Genetics Computer Group (Madison, WI) sequence analysis programs (17). Chromosomal Gene Laliation. A panel of 24 Chinese hamster ovary (CHO)/human somatic cell hybrids were used to localize the HBGR1 and HBGR2 clone genes to human chromosomes with Southern blot analysis. The mapping panel consists of hybrid cell lines containing a derivative human chromosome 5 and one or two additional chromosomes. The parental CHO cell line used to construct most of the hybrids, UCW56, harbors a LARS gene mutation encoding a temperature-sensitive leucyl-tRNA synthetase (18). The temperature-sensitive phenotype is complemented by the human LARS gene product, thus allowing selection at 39'C of hybrids containing human chromosome 5. Four additional hybrids were included that used other modes of selection, specific for the retention of chromosome X, 12, or 18. Cell line HHW1164 is a subclone of UV24HL5 (Larry Thompson, Lawrence Livermore National Laboratory). Each hybrid was characterized by trypsin-Giemsa banding and G-11 staining. Together, the 24 hybrids allow for the assignment of probes to any one of the 24 distinct human chromosomes. DNA from these 24 cell hybrids, CHO cells, and HeLa cells was digested with EcoRI or HindIII and blotted as described (19), except that the transfer membrane was'Genatran (Plasco, Woburn, MA). Two probes, GR1A from clone HBGR1 and GR2A from clone HBGR2, were used for Southern analysis: probe GR1A contains the first 452 base pairs (bp) of the HBGR1 5' end (-142 to 310); probe GR2A contains =900 bp of 3'-untranslated region from clone HBGR2, from a Xho I site to the far 3' end. In addition, a 600-bp probe, GR1B, from the HBGR1 3' end (2208-2808) was used for subchromosomal assignment. Functional Expression in Xenopus Oocytes. cRNA was synthesized directly from the HBGR1 clone in the SK(-) vector by using T3 'RNA polymerase (20, 21). Oocytes were removed from anesthetized adult female Xenopus laevis (Nasco, Fort Atkinson, WI) and dissociated, as described (22-25). Isolated follicule-free stage V and VI oocytes were injected (50 nl) with cRNA dissolved in distilled water at 0.4 mg/ml and maintained at 20°C in Barth's medium (22-25). Oocytes were transferred to the recording chamber (vol %0.2 ml) continuously perfused (=2 ml/min) with frog Ringer's solution (115 mM NaCl/2.5 mM KCl/2 mM CaCl2/5 mM glucose/5 mM [N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid, pH 7.4], in the presence or absence of tested ligands. Whole-cell currents were measured under voltage clamp (Turbo TEC 01C, NPI Electronics, Tamm, F.R.G.) with the conventional two microelectrodes (0.7-3 Mfl in 3 M KCl). Currents were elicited at a holding potential of -120 mV, unless specified otherwise. Only oocytes with a resting potential of c-30 mV were studied. All experiments were done at 22 ± 2°C. Records were filtered at 50 Hz and digitized at 100 Hz by using an Axon TL-1 interface (Axon Instruments, Burlingame, CA) connected to a 386based Everex Step 386 computer (Everex, Fremont, CA) computer. Axotape and pClamp 5.5 packages (Axon Instruments) were used for data acquisition and analysis. RESULTS AND DISCUSSION Primary Sequence. Hybridization screening at high stringency and subsequent plaque purification yielded four positive clones. Restriction digests suggested that two clones are from one transcript, whereas the other two are from a different transcript. One clone, HBGR1, is 3 kb in length and contains a full-length glutamate receptor cDNA highly ho-

Proc. Natl. Acad. Sci. USA 89 (1992)

mologous to the rat brain clone GluRl (7) or the flop version of the GluR-A clone (8, 26). HBGR1 contains about 140 bp of 5'-untranslated regions and 80 bp of 3'-untranslated region without poly(A) or polyadenylylation signal sequences. Another clone, HBGR2, is 2.9 kb in length and contains twothirds of the coding region of a gene homologous to clone GluR2 or GluR-B (7, 8). The sequences from clones HBGR1 and GluRl share 90%o identity at the nucleotide level and 99o homology at the amino acid level. Within the 140 bp of 5'-untranslated regions, the nucleotide sequences between the two cDNAs do not diverge widely. Fig. 1 shows the deduced amino acid sequence of the HBGR1 cDNA. Differences between the HBGR1 and GluRl proteins are shown in boldface type. Most changes are conservative and occur in the N-terminal half of the sequence. Clone HBGR1 codes for a 906-residue protein with estimated Mr -100,000. A hydrophobicity plot of the HBGR1 polypeptide displays six hydrophobic stretches, four of which are long enough to traverse the membrane bilayer as a-helices. These four segments, identified as M1-M4, are underlined. M2 is amphipathic and is, therefore, a plausible candidate for the segment that forms the channel in an oligomeric protein. Residues S-572, S-576, A-579, and Q-583 would be exposed to the channel lumen. The M2 segment is flanked at the N and C termini by a glutamate and an aspartate. The location of these negatively charged residues at the presumed entry and exit of the pore could generate a surface enrichment of cations. Accordingly, these residues may contribute to the cationselectivity of the channel. Recent experimental evidence supports the notion that M2 segments play an important role in determining the selectivity and the current-voltage (I-V) characteristics of kainate receptors (27). The N-terminal segment preceding Ml contains all potential N-glycosylation sites (amino acids 45, 231, 239, 345, 383, MQHIFAFF CTGFLGAVVG

ANFPNNIQIG LTEPPKLLPQ SKGVYAIFGF ITPSFPVDTS HYKWQKFVYI NWQVTAVNIL LVVVDCESER

ILANLGFMDI TDTIPAKIMQ TSALTYDGVK NAGDCLANPA LTGNVQFNEK IGYWNEDDKF YIVTTILEDP

CVELAAEIAK PDTKAWNGMV REEVIDFSKP

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GLFPNQQSQE HAAFRFALSQ IDIVNISDSF EMTYRFCSQF YERRTVNMLT SFCGALHVCF

-1 30 60 90 120 150 180 210 240 270 300 330 360 390 420

NQFVLQLRPE LQDALISIID YDADRGLSVL QKVLDTAAEK TTTEEGYRML FQDLEKKKER LNAILGQIIK LEKNGIGYHY DLNKFKESGA NVTGFQLVNY QWKNSDARDH TRVDWKRPKY VMAEAFQSLR RQRIDISRRG VPWGQGIDIQ RALQQVRFEG GRRTNYTLHV IEMKHDSIRK VPAATDAQAG GDNSSVQNRT YVMLKKNANQ FEGNDRYEGY HVGYSYRLEI VSDGKYGARD 450 GELVYGRADV AVAPLTITLV 480 FMSLGISIMI KKPQKSKPGV 510 IWMCIVFAYI GVSVVLFLVS 540 EFEEGRDQTT SDQSNEGIFE 570 MOOGCDISPR SLSGRIVGGV 600

WWFFTLIIIS SYTANLAAFL TVERMVSPIE 630 SAEDLAKQTE IAYGTLEAGS TKEFFRRSKI AVFEKMWTYM KSAEPSVFVR TTEEGMIRVR KSKGKYAYLL ESTMNEYIEQ RKPCDTMKVG GNLDSKGYGI ATPKGSALRN PVNLAVLKLN EQGLLDKLKN KWWYDKGECG SGGGDSKDKT SALSLSNVAG CYKSRSESKR LPRNSGAGAS SIPCMSHSSG

660 690

720 750 780

VFYILIGGLG LAMLVALIEF 810 MKGFCLIPQQ SINEAIRTST 840 SGGSGENGRV VSHDFPKSMQ 870 .888

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FIG. 1. The deduced amino acid sequence of the HBGR1 cDNA. The human receptor is one glycine shorter than the rat receptor GluR1 (amino acid 851). Amino acid differences between the HBGR1 and GluRl proteins are shown in boldface type. These are as follows: H-2 Y.S-49 T.D113 E K-126 T.K-142 R. Q-1 --,P. 1-199 -- V, A-230 -* R, K-247 -- R, K-253 -* R, N-254 -* T, A-257 -+ S, S-357 -- G, S-771 -. T, S-851 -* G, and H-863 -- Q for HBGR1 and GluRl. Potential transmembrane segments (M1-M4) are underlined. --.

,

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Proc. Natl. Acad. Sci. USA 89 (1992)

1445

that carry chromosome 4 or 8. The primer set amplified a 250-bp fragment with DNA isolated from HeLa cells and HHW416 cells, which carry chromosome 4. In contrast, the primer set failed to amplify the 250-bp fragment using DNA isolated from UCW104, the hamster cell line, and from HHW 811, HHW509, and HHW867, hybrids that carry human chromosome 8. PCR, therefore, assigns the GR2A probe to chromosome 4. To refine the localization of GR2A probe, a panel of seven additional cell hybrids containing various deletions of chromosome 4 was used (Fig. 2B). Taken together, the results map clone HBGR2 to chromosome 4q2534.3 and suggest that chromosome 8 carries a pseudogene or related gene. Functional Expression of HBGR1 Protein in Xenopus Oocytes. The pharmacological properties of the glutamate receptor channel encoded by the cDNA clone HBGR1 were characterized after expression of cRNA in Xenopus oocytes. HBGR1 cDNA codes for a glutamate receptor of the kainate subtype and not of the NMDA subtype, as illustrated in Fig. 3. The figure shows the time course of change in I recorded at an applied V = -120 mV, upon perfusion of the oocyte with the agonist kainate and/or other agonists or antagonists (5), according to the pulse sequences shown below the current traces. As displayed in Fig. 3A, perfusion with buffer containing 0.1 mM kainate evokes an inward current that activates slowly and does not desensitize. As the agonist is washed out with Ringer's solution (second kainate pulse OFF) or with Ringer's solution containing 0.1 mM NMDA, the current returns to the initial baseline. Perfusion with Ringer's solution containing 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a competitive antagonist of non-NMDA receptors, collapses the current elicited by the second kainate pulse and

and 388), assigning this segment to an extracellular domain of the protein. The loop connecting the postulated M3 and M4 segments contains consensus sites for phosphorylation by distinct protein kinases: cAMP-dependent kinase (amino acid 658), protein C kinase (amino acid 692), and tyrosine kinase (amino acid 707), restricting this stretch to an intracellular domain. These considerations suggest a protein model for the human brain glutamate receptor consisting of four membrane-spanning segments with N and C termini exposed to the extracellular surface of the membrane. The HBGR1 protein, thus, exhibits the structural features postulated for other glutamate receptors (6-9, 26-28). Mapping of Human Brain Glutamate Receptors to Human Chromosomes. The results of Southern analysis on a somatic cell-hybrid mapping panel are summarized in Table 1. Plus signs indicate presence of the human chromosome in the corresponding cell line; asterisks indicate a positive signal on the Southern blot using the GR1A or GR2A probe. A discordance value of zero signifies that the probe is most likely present on that chromosome. The mapping panel assigns clone HBGR1 to chromosome 5. A panel of seven additional somatic cell hybrids and the GR1B probe was used to sublocalize clone HBGR1 on chromosome 5. Fig. 2A shows an ideogram of chromosome 5 and the eight cell lines that carry the various deletions. The GR1B probe was present in all cell lines, assigning clone HBGR1 to the region of overlay-namely, 5q31.3-33.3. The GR2A probe hybridized to a 1-kb band (GR2A.1) on chromosome 8 and to a 2-kb (GR2A.2) band on chromosome 4 (Table 1). To clarify the ambiguity, a set of PCR primers designed to amplify a 250-bp fragment from the 3'-noncoding region of clone HBGR2 was used to screen hybrid cell lines

Table 1. Localization of the GR1A and GR2A probes to human chromosomes using a somatic cell hybrid mapping panel Cell

Line CHO 1144

1164

PROBE ORI4OSOME -_1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y GR1A GR2A.1 GR2A.2 --

890 1125 967

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811. 3241113 690 711 441 1107

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4844 983

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342 416 105 1126 509

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4

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17 4 2

17 4 2

17 18 2 2 2 3

18 17 4 4 2 2

4

4

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17 17 17 18 17 4 2

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4 2

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4 2

Panel of 24 somatic cell hybrids used to map the GRIA and GR2A probes. Plus signs (+) indicate that the particular hybrid cell line carries the corresponding human chromosomes. HHW484 carries only the p arm of chromosome 12. Asterisks (*) indicate that the probe hybridized to DNA from the corresponding cell line. Question marks (?) indicate questionable assignment due to the lack of resolution between hamster and human bands on the Southern blot. The discordance value for a given chromosome number and probe or band is calculated by adding the number of cell lines that carry the chromosome but show no hybridization band and the number of cell lines that do not carry the chromosome but hybridize with the probe. A zero discordance value signifies a perfect match between presence of a chromosome in a given cell line and identification of the probe location to the given cell line and, therefore, assigns the probe to the corresponding chromosome. The GR1A probe was assigned to chromosome 5. The GR2A probe hybridized to a 1-kb band (GR2A.1) on chromosome 8 and a 2-kb band (GR2A.2) on chromosome 4. The exact assignment of GR2A was confirmed by PCR (see text). CHO, Chinese hamster ovary cell line 104.

tDiscordance.

1446

Neurobiology: Sun et al.

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Proc. Natl. Acad. Sci. USA 89 (1992)

0.1 mM KAINA 0.1 mM NMDA 10 uM CNQX AGONIST/ANTAGON

ON -LOFF ON OFF

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-

0m AN

0.1I mKl KINAT

4

FIG. 2. Localization of the HBGR1-encoding gene on human chromosome 5q31.3-33.3 and the HBGR2-encoding gene on chromosome 4q25-34.3. (A) Ideogram of G-banded chromosome 5, with solid lines depicting the portion of chromosome 5 in the indicated hybrid cell line. The dotted lines for HHW213 indicate that probes for these regions were determined present by PCR and/or hybridization; however, the exact breakpoints of the interstitial deletions are not known. The presence of the GR1B probe in all eight cell lines constrains the assignment of HBGR1 to the region of overlays, 5q31.3-33.3. (B) Ideogram of G-banded chromosome 4 and a bar representation of eight different somatic cell hybrids with indicated deletions. Presence (+) or absence (-) of the GR2A probe localizes HBGR2 to 4q25-34.3.

prevents further current generation as long as it remains in the perfusion buffer (agonist and antagonist pulses ON). As the antagonist is washed out of the chamber (antagonist pulse OFF), an inward current is elicited; the current returns to the original baseline at the cessation of the agonist pulse. These cyclic responses can be elicited virtually indefinitely by repeating the agonist/antagonist pulse sequences illustrated in Fig. 3A and, therefore, are used as a pharmacological assay for agonist and antagonist specificity; this result is illustrated in Figs. 3 B and C. The activity of several synthetic and endogenous agonists is shown in B. Domoate mimics the kainate-evoked signals; in contrast, quisqualate, AMPA, and L-glutamate do not elicit current responses at equivalent concentrations (0.1 mM). As displayed in Fig. 3C, the kainate-activated current is specifically and reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, but is insensitive to 2-amino-5-phosphonovalerate or kynurenic acid (or a combination of both), two selective antagonists of the NMDA receptor subtype. Thus, the current responses generated in oocytes expressing the HBGR1 gene product display the agonist specificity and antagonist sensitivity characteristic of a glutamate receptor of the kainate subtype. To assess the specificity of the receptors encoded by HBGR1, the dose-response characteristics for the higher affinity agonists, kainate and domoate, were measured (Fig. 4A). The agonist concentrations at which the current amplitudes were. 50% of the maximal responses (EC50) were 1.9 + 0.3 uM (n = 2) for domoate and 45 + 3 AuM (n = 4) for kainate and were best-fitted with Hill coefficients of 1.2 ± 0.4 and 0.9 ± 0.1. The current responses elicited by other agonists at submillimolar concentrations were of significantly lower amplitude. The relative efficacy of different agonists measured at a fixed concentration of 0.1 mM was as follows: domoate > kainate >> AMPA 2 quisqualate 2 L-glutamate. This ranking is consistent with the notion that HBGR1 is a kainate-specific glutamate receptor. The highly homologous rat brain clone GluR-A, however, forms an AMPA-selective

OFNF ON

0.1 mM DOMOATJL 0.1 mM AGONIST

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30 s

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0.1 mM KAINATEJ I mM APV+ O.1 mM

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258

FIG. 3. The HBGR1 gene product is a kainate-selective glutamate receptor. Current responses activated by glutamate receptor agonists in Xenopus oocytes expressing RNA transcripts of the HBGR1 cDNA. Recordings obtained 2-5 days after injection of 20 ng of HBGR1 mRNA. V = -120 mV. Oocytes were perfused with Ringer's solution supplemented with agonists and/or antagonists, as indicated; ON and OFF denote initiation and cessation of perfusion pulses. For display, records were filtered at 5 Hz. A downward deflection indicates inward current flow. The figure illustrates representative results offive independent determinations. CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; QUIS, quisqualate; KYN, kynurenic acid; APV, 2-amino-5-phosphonovalerate.

glutamate receptor (8, 26, 28). AMPA, quisqualate, and L-glutamate elicit biphasic currents that exhibit a fast desensitizing and a steady-state component (26, 29). Our recording conditions allow us to resolve only the latter component and, therefore, a transient initial desensitization cannot be ruled out. Hence, it is likely that clone HBGR1 codes for a kainate/AMPA-selective glutamate receptor cognate to members of the glutamate receptor family from rat brain. The I-V relationships for kainate- and L-glutamateactivated channels are shown in Fig. 4B. The I-V relations for both high- and low-affinity agonists display a conspicuous rectification. At V more positive than -40 mV, the currents are practically undetected. These features match those recorded from homooligomeric GluRl glutamate receptors expressed in oocytes (7, 28). The nature of the asymmetric I-V relations is unclear. However, it is known that coexpression of GluR2 protein with either GluRi or GluR3 protein (28) or combinations of GluR-B protein with either GluR-A or GluR-D protein (27) generate receptors with agonistactivated responses that exhibit linear I-V characteristics. This mechanism may contribute to the diversity of glutamate receptors, as inferred from electrophysiological and pharmacological studies, although other factors such as divalent-ion channel blockade, voltage-dependent properties, or heterologous expression may be involved. It is worth noting that

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Proc. Natl. Acad. Sci. USA 89 (1992)

A zE-w

1.0

PQ

N 0.5 -!!

A: 0

z

0.0

10-61 i0-5 10-4 10-3 CONCENTRATION (M)

10-7

B

0.25

VOLTAGE (mV) -140

E-

-100

-60

-20

r20

z 04

L-GLU --0.5 N

KAINATE A: 0

--1.0

FIG. 4. Dose-response and I-V relationships of HBGR1 expressed in oocytes. (A) Dose-response curves. Domoate (e)- and kainate (v)-activated currents at V = -120 mV. Each point represents the mean SD of agonist-activated currents collected from two oocytes for domoate and four oocytes for kainate. Solid lines depict theoretical fits to a Michaelis-Menten binding isotherm given by III.. = K[agonist]'/(l + K[agonist]Y), where I is the steady-state current amplitude at a given agonist concentration, n is the number of ligand-binding sites, and K is the affinity constant for the agonist concentration at which response is half-maximal (I.,a). I,,. was derived from the y intercept of the best-fit to a 1/I vs. 1/[agonist] function. (B) I-V characteristics of 100 1sM kainate- and 1 mM L-glutamate-activated currents. Currents were normalized for both ligands with respect to the steady-state current elicited by 100 AM kainate at V = -120 mV. Number of oocytes equals 2. ±

cultured hippocampal neurons express populations of kainate-activated channels with both linear and rectifying properties (30), suggesting the in vivo occurrence of homo- and heterooligomeric assemblies. Note. After submission of this manuscript, a paper appeared reporting the cloning of a human cDNA encoding a glutamate receptor, identified as GluHi (31). This cDNA shows differences with HBGR1 primarily in a stretch that precedes the presumed transmembrane segment M4, in a region corresponding to the alternatively spliced exon identified in the rodent clones by Sommer et al. (26) and designated as flip and flop forms of GluRl. Our clone HBGR1 corresponds to the flop version, whereas that reported by Puckett et al. (31) corresponds to the flip form. GluHl mapped to 5q33 (31), in accord with our results for HBGR1.

We thank D. Ware for participation in the construction of the cDNA library, D. Q. Hoang for DNA sequencing, J. Tomich and B. Cai for oligonucleotides, R. Miledi for assistance in setting up the oocyte system, J. Furse for invaluable assistance, and members of our group for perceptive comments. This work was supported by grants from the National Institutes of Health (MH-44638 to M.M. and GM33868 and HG00047 to G.A.E.), the Department of the Army Medical Research (DAMD 17-89-C-9032 to M.M.), and by a Re-

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