THE JOURNAL OF BIOLOGICAL. CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 36, Issue of December 25, PP. 22317-22320,199O Printed in U.S. A.

Identification and Functional Expression of a Novel Ligand Binding Subunit of the Inhibitory Glycine Receptor*

(Received for publication, July 5, 1990)

Jochen Kuhse, Volker Schmieden, and Heinrich Bet4 From the Zentrum fiir Molekulare BioloEie Heidelberg, Universitiit Heidelberg, Im Neuenheimer Feld 282, 6900 Heidelberg, Federal Republic of Germany

By homology screening of a rat brain cDNA library we have identified a novel (Y subunit variant of the inhibitory glycine receptor, cr3. The deduced protein exhibits 82-83% amino acid identity to the previously characterized rat and human cwl and cr2 subunit se- quences. Upon heterologous expression in Xenopus lae- vis oocytes, the (~3 subunit formed functional agonist- gated chloride channels which displayed a low affinity for glycine and only small responses to taurine. Ampli- fication of a3 transcripts by polymerase chain reaction revealed high levels in spinal cord at later postnatal stages of development. These data indicate CY subunit heterogeneity of the inhibitory glycine receptor in adult rats that may result in pharmacologically distinct receptor subtypes.

Heterogeneity of neurotransmitter receptors is thought to play an important role in diversifying interneuronal signaling in the nervous system. The identification and characterization of the molecular components of these receptors therefore constitutes a basic step for understanding the biology of neuronal communication. Moreover, the identification of ho- mologous receptor subunits displaying different functional properties may provide clues to important structure-function relationships. Thus, cloning of receptor variants has become a major goal of recent molecular biology approaches in neu- robiology (1, 2).

The inhibitory glycine receptor (GlyR)’ is a ligand-gated chloride channel protein which mediates inhibition of neu- ronal activity in spinal cord and other regions of the vertebrate central nervous system (3). Activation of the GlyR by the agonists glycine, p-alanine, or taurine causes hyperpolariza- tion of the postsynaptic membrane that is readily blocked by the high affinity antagonist strychnine, an alkaloid from Strychnos nux-vomica (4, 5). The receptor has been purified by affinity chromatography and shown to represent a penta- mer composed of ligand binding subunits of 48 kDa (a) and homologous polypeptides of 58 kDa (/3) (6-10). Recently, the

* This work was supported by Deutsche Forschungsgemeinschaft (SFB 317 and Leibniz Programm), Bundesministerium fiir Forschung and Technologie (BCT 365/l), and Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank’L‘M/EMBL Data Bank with accession number(s) M38385.

$ Present address: Max-Planck-Institut fiir Hirnforschung, Deut- schordenstrasse 46, D-6000 Frankfurt 71, FRG.

1 The abbreviations used are: GlyR, inhibitory glycine receptor; PCR, polymerase chain reaction; kb, kilobase(

primary structures of these GlyR subunits (al and p) have been determined by cDNA sequencing (11, 12) and shown to define a receptor superfamily that includes nicotinic acetyl- choline and GABA* receptors (13). Furthermore, (Y subunit variants have been identified in human ((r2) and rat (a2*) which are thought to represent ligand-binding subunits of a neonatally expressed GlyR isoform (14, 15). This neonatal receptor displays only low strychnine binding affinity and contains an (Y subunit of apparent molecular mass of 49 kDa (16, 17).

Heterologous expression in Xenopus oocytes or mammalian cell lines indicates that GlyR (Y subunits are capable of assem- bling into functional agonist-gated chloride channels with ligand affinities similar to those of the receptor detected in viuo (18, 19). Injection of in uitro-synthesized cy subunit mRNAs into Xenopus oocytes therefore has been exploited to analyze the pharmacology of individual subunit variants, and significant functional differences between al, ~2, and a2* polypeptides have been disclosed by this approach (14, 15).

Here we describe the identification and characterization of a novel GlyR (Y subunit variant, termed cr3, which is expressed in adult spinal cord and generates chloride channels of low glycine affinity upon expression in oocytes. Our data demon- strate GlyR 01 subunit heterogeneity in the adult rat central nervous system.

MATERIALS AND METHODS

Isolation of ClyR a3 Subunit cDNAs-A rat brain cDNA library (5 X lo5 plaque-forming units) (20) was screened under low stringency conditions using as a probe a 32P-labeled (0.8 x 10s cpm/pg DNA) 1.4-kb EcoRI fragment of a rat GlyR or1 cDNA construct (18). Hybridization was done in 20% (v/v) formamide, 750 mM NaCl, 75 mM Tris-HCl, pH 7.5, 12.5 mM EDTA, 0.1% (w/v) sodium dodecyl sulfate, and 0.1% (w/v), each, of bovine serum albumin, Ficoll 400, polyvinylpyrrolidone, and 10 fig/ml denaturated herring sperm DNA at 42 “C for 18 h. Washing of nitrocellulose filters was performed three times in 2 X SSC (300 mM NaCl, 30 mM sodium citrate) at 55 “C for 15-20 min. In addition, replica filters obtained from the same plates (21) were hybridized under stringent conditions to a radiolabeled cDNA probe containing part of the coding sequence (base pairs 630-1250) of the GlyR @ subunit cDNA (12). From clones positive with the al probe only, overlapping DNA restriction frag- ments were subcloned into pSPT19 (Boehringer Mannheim), and both strands were sequenced using the chain termination method (22). In addition, different synthetic oligonucleotides served as se- quencing primers using recombinant plasmid DNA (pGR48n3) as a template. This clone contained the full-length EcoRI fragment iso- lated from purified X DNA in pSPT19.

Expression of a3 Sequences in Xenopus Oocytes-Recombinant plasmid DNA (pGR48a3) was linearized with SacI, and in vitro transcription was carried out using a transcription kit (Boehringer Mannheim). Oocytes were obtained from anesthetized adult Xenopus laeuis, and injection of cRNA and electrophysiological recordings were performed as described previously (18).

Amplification of Transcripts by Polymeruse Chain Reaction (PCR)- RNA was extracted from rat spinal cord and brain cortex by the

22317

Glycine Receptor a3 Subunit

method of Cathala et al. (23). Poly(A)+ RNA was enriched by chro- matography on oligo(dT)-cellulose. For PCR (24), poly(A)+ RNA (2 pg) was reversely transcribed into cDNA with 30 units of avian myeloblastosis virus reverse transcriptase. About 100 ng of cDNA were used as template for PCR in a volume of 100 pl of 10 mM Tris- HCI, pH 8.3, containing 200 pM of each deoxynucleoside triphosphate, 50 mM KCl, 2.5 mM MgCL, 0.001% (w/v) gelatin, and 200 pmol of each primer oligonucleotide. Sequences of synthetic oligonucleotides used to amplify (~3 sequences were located at positions 282-303 (sense) and 609-660 (antisense) of the cDNA sequence (Fig. 1). For (~1 amplification, corresponding sense and antisense oligonucleotides covered positions 426-447 and 727-748, and for c~2* amplification positions 288-308 and 868-890, of the respective cDNAs (11, 15). 2.5 units of Ampli Taq (Perkin-Elmer Cetus Instruments) were present per reaction under the following cycle conditions: denaturation at 95 “C for 0.8 min, annealing at 55 “C for 0.8 min, and DNA synthesis at 72 “C for 1.5 min. After 26 cycles, 15 ~1 of each reaction were separated on 1% agarose gels. After blotting onto nylon membranes with 20 x SSC, the filters were hybridized with the following radio- labeled DNA probes of various rat GlyR subunits: a 1.4.kb oil EcoRI fragment as described (18), the 2.3-kb EcoRI fragment of clone pGR48n2’ (15), and the 2.4-kb EcoRI fragment of clone pGR48a3. Control experiments using different cycle numbers for amplification and PCR with cloned cDNAs showed that this method allowed comparison of relative mRNA levels of a given e-subunit sequence. To control for the amount of cDNA used at different developmental stages, quantitation of oil and 012* mRNAs by PCR was compared with Northern analysis and S, nuclease protection data and found to strongly correlate with the latter results (not shown).

RESULTS AND DISCUSSION

To identify subunit variants of the GlyR, we performed homology screening of a rat brain cDNA library. From 23 clones hybridizing with an (~1 probe under low stringency conditions, two strongly hybridizing plaques were not detected with a p subunit DNA probe and, therefore, were character- ized further. Partial sequence analysis of one of these clones (pGR48-14) revealed that it encoded a novel homologous o( subunit but lacked the coding sequence of the N-terminal part of the protein. To isolate a full-length cDNA clone, the XgtlO library (10s plaque-forming units) was rescreened under stringent conditions with the radiolabeled EcoRI fragment of clone pGR48-14 and a 50-mer oligonucleotide containing a sequence from the 5’ end of clone pGR48-14. Out of 11 clones hybridizing with the radiolabeled EcoRI fragment, we isolated two identical full-length clones with EcoRI insert fragments of about 2.4 kb. Analysis of the DNA sequence of one of these clones revealed an open reading frame encoding a putative polypeptide of 464 amino acids (Fig. 1) which displays high homology to the al and ot2 GlyR sequences from rat and human (11, 14). In front of an ATG at position 400 a second potential translation start codon is located in-frame at posi- tion 351. However, comparison of the nucleotide sequences surrounding both ATGs suggested that the ATG at position 400 is the translational start codon of a3 because only this codon fulfills the criteria of an eukaryotic translational start site (25). Processing of the polypeptide at the predicted signal peptide cleavage site indicated in Fig. 1 results in a mature protein of 431 amino acids with a calculated molecular mass of 49.892 Da and a theoretical isoelectric point of 8.45. Align- ment of the novel deduced amino acid sequence, a3, to the other known (Y subunits from rat revealed sequence identities of 82 and 83% with a2* and al, respectively (Fig. 2). The putative transmembrane regions Ml-M4 as well as the extra- cellular region of the mature proteins are highly homologous. This part of the cu3 subunit shares 88-91% sequence identity with the c~2 and al sequences. Considerable sequence diver- gence is found in the putative cytoplasmic region between M3 and M4 of the GlyR polypeptides. Here homology is only around 54-57%; however, high conservation resumes in front

1 61

121 181 241 301 361

CTGTTGTGCTTCCTCGATCCCTGCCTGGGCCAGAGCCGAGGAGTCCTTTTCTG CTGATAGAACTGTGRGGAACTGTTGCTCAGGACCTCTGCAA GTCTCTCGCTTTTCTGTGGCTTCACAGTWLAATCACTCGCT CCTTCTGCTCCAGTGCTCAGAGGAGGGGTCTAGGAAAACATCAAGTGTTTGAAAGAGGGA TTCGGRGTACTGCCGGAGCTGTCTGAGGTGCTGCTTCA GCCCAGAAGGGGTTAGATGGACGTTTTCACTTCTT~GTCTTTCTGGTCCCCCATGCCTTGG ATAAGACTGTTTTULGGATCGGGAATATCTTTCCGTATCATG~CCACGTGAGACAC~T

HAHVRHF

421

481

541

ACGARGGARRVLARCA~G~C~T~~~-~T~~~~~T~~CACCTTCT~TTTT~~~ TKETNSARSRSAPMSPSDFL

1 GACARACTAATGGGGAGGACATCCGGGTATGATGCAAGAAGGT

DKLMGRTSGYDARIRPNFKG

601

661

CCTCULGTPRATGTCA~T~~~CAACATATTCATARACAGCTC~~~~~~~~~~~~ P P "p?J" TCNIFINSFGSIAET

ACTATGGATTACAGAGTAAACATTTTCCTTCGTCAGAAGT~G~GTGG~T~TCCTCGCCTT~A TMDYRVNIFLRQKWNDPRLA

721 TACAGTGAATACCCTGATGATTCATTAGACCTCGACCCCATCCATGTTG~CTCCATATGG YSEYPDDSLDLDPSHLDSIW

781 MACCTGACTTGTTCTTTGCTAATGAGAAGGGGCTAACTAACTTCCACGAAGTCACCACCGAT KPDLFFANEKGANFHEVTTD

841 AACAAGCTGCTAAGAATTTTCAAAAATGGAAATGTTCTTTATTCAATAAGGCTGACATTA NKLLRIFKNGNVLYSIRLTL

901 ACACTCTCTTGTCCAATGGATCTCAAGAATTTTCCCAATG~TGTTCAAACATGCATAATG TLSCPMDLKNFPMDVQTCIM

961 CAACTCGAAAGCTTTGGGTACACGATGAATGATTTGCA QLESFGYTMNDLIFEWQDEA

1021 CCAGTACAAGTGGCTGAAGGCTCACTTTGCCTCAATTCT PVQVAEGLTLPQFLLKEEKD

1081 TTGCWLTACTGCACTRAACACTACAATACAGGAAAGTTTACATGCATA~GTACGATTT LRYCTKHYNTGKFTCIEVRF

1141 CATCTTGAGCGGCAAATGGGTACTACTTGATCULGATGTCCTTCTGATT HLERQMGYYLIQMYIPSLLI

1201 GTCATTCTGTCCTGGGTCTCTTCTGGATTARCATGWLTGTCGGGTAGCG "1LSW"SFWINMDAAPAR"A. .254

1261 TTGGGTATCACCACTGTACTTAC~T~CCACG~GAGTTTTCTTTA L G I Ti" L T MT T Q S S G S R A S L 274

1321 CCARAGGTGTCCTATGTCAAGGCAATTGACATTTGGATGGCAGTGTGTCTCCTTTTTGTG P KVS YVKA I D I WMAVC L LF V 294

1381 TTCTCAGCACTTCTGGAGTATGCAGCCGTGAATTTTGTATCAAGGCAACACAAA~CTG F SALL EYAAVN F" S RQ H K E L 314

1441 CTGAGGTTTCGGCGAAAGAGGAAAAATAAAACAGAAGCTTTTGCACTGGAGAAGTTTTAC L R F R RK R KN KT EAF A L E K F Y 334

1501 CGTTTCTCAGACACGGATGATGAGGTGAGGGAGAGAGTCGGCTCAGCTTCACT~CTATGGA RF SD T D D E" R E S R L S F T A Y G 354

1561 ATGGGGCCCTGTCTTCARGCAARGGATGGTGTGGTTCCAATGCTGTC I4 G P C LO AK D G "VP KG P NH A" 374

1621 CAGGTCATGCCAAAGAGCGCCGATGAAATGAGGAAGGTCTTCATCGACAGGKTAAGAAG QVH P K SAD E MRK" F I D RA K K 394

1681 ATCGACACCATCTCCCGAGCCTGCTTTTCCGCATTTCTTTCTAC I D T I S RA C F P LA F L I F N I F Y 414

1141 TGGGTTATCTATAAAATCCTAGGCATGAAGACATTCATCATCAGCAAGATTAAGTCTAT W"IYKILRHEDIHHQQD 431

1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401

GAAGGCATACAAAAACAGAGAT-TGAG&AGWAGAGTCCCGCTAAAGGAGCGGGGTGTGT GTGCATGTGGGGTGCAGGTGGAGTCAGTGCATCAGAACTTTA TTTTGATTTCCTATGTAAAATAGTGAGAVLGTTTGGTTTGGTT-TGATACAAAATGTACATcG TGGGTGTCCTGCTTCATARTCCTGACAGGTGATCTGTGATCTGTTGTCTTCCTAGTGT-TATG~G TTGCATATGCTTTACAAGCACATAGAGTAGACATGATARCT TTTAATTCAATTATAGTAAACACTGCGGAAGCAAGATTTACACAATAAAATGTATAAAGC AAGTATAGCAARATGATGAGTATRAAATGTCTTGTCTT~GATATACAGCATATCGATG~G TAACAAGATCATTAAATGTUGGTCACTCATGTCAGAACACTGTCCATAGAAACAGTGT GGAACCAACAACCACAATAAAAACAAAACAAAATCGGATGGTC~TGAAAGCCATCATTTAGA AUICTATGGCTTAAGCRATCTGTTTAT-TTCTACTTATCTTTACATCTCTAGG~T GAGGAGGAAGATG 2413

1. Nucleotide and deduced amino acid sequences of the

-27

-7

14

34

54

74

94

114

134

154

L74

194

214

234

GlyR (~3 subunit from rat. The deduced amino acid sequence of the encoded polypeptide is indicated in the single letter code below the nucleotide sequence. The putative signal peptidase cleavage site is indicated by an arrow. Proposed transmembrane spanning regions Ml to M4 are underlined, and a putative extracellular N-glycosylation site is boxed. Numbering of nucleotides is indicated on the left, and of amino acid residues on the right side of the figure.

Glycine Receptor a3 Subunit

a1 17 LMGRTSGYDARIRPNFKGPPVN~NIFINSFGSIAETTMDYRVNIFLRQ a2* 24 LMGRTSGYDARIRPNFKGPPVNVTCNIFINSFG~TTMDYRVNIFLRQ a3 17 LMGRTSGYDARIRPNFKGPPVElVTCNIFINSFGSIAETT*R"~~~LRQ

A~YPDDSLDLDPSMLDSIWKPDLFFANEKG~ AYSEYPDDSLDLDPSMLDSIWKPDLFFANEKGANF

* a1 117 LLRI GNVLYSIRITLT~PMDLKNFPMDVQT~~MQLESFGYTM~L ai?* LLRISKN~YSIRLTLTLSCPMDLKNFPMDVQT~QLES~~~DL a3 117 LLRI~NGNVLYSIRLTLTLSCPMDLKNFPMDVQTCIMOLESFGYTMNDL

a1 167 IFEW a2*174 IFE S

~Q~~~F;LTLPQF~LKEEK~LR~~T~"~~TG~~T~~

EC4

PVQVAEGLTLPQFILKEE~CTKHYNTGKFTCI 0.3 167 IFEW E "QVAEGLTLPQ KEEKDLRYCTKHYNTGKFTCIEVRFHL

al 217 ERQMGVYLIQMYIPSLLIVILSYI)SFWINMDAAPAR~GITTVLTMTTQ C&2*224 ERQMGYYLIQMYIPSLLI"ILSW"SFWINMDAAPAR"ALGITT"LTMTTQ Cr.3 217 ERQMGYYLIQMYIPSLLI"ILSWVSFWINMDAAPARVALGITT"LT~TTQ

- al 267 SSGSRASLPKVSYVKAIDIWMAVCLLFVFSALLEYAAVNFVSRQHKELLR a2*274 SSGSRASLPKVSYVKAIDIWMAVCLLF~LLEYAAVNFVSRQHK~LR U3 267 SSGSRASLPKVSYVKAIDIWMAVCLLFVFSALLEYAAVNFVSRQHKELLR

FIG. 2. Alignment of rat al, aZ*, and a3 polypeptide se- quences. Numbering starts with the proposed N-terminal amino acid of the mature proteins. Identical residues are indicated by solid boxes. Gaps were introduced to maximize identical sequence positions. Po- tential signal cleavage sites are indicated by arrows. Proposed disul- fide-bonded loop regions are marked by asterisks, and the putative membrane spanning regions Ml to M4 by solid lines.

A B 0.1 0.5 4mM -- -

$:

I i

-;r

2 ,500

100 nM Stzy

5 If Iii

z5i $ ‘1:

0

-’ log &or& (mM;

FIG. 3. Expression of the rat (~3 subunit in Xenopus oocytes. RNA transcribed in vitro from pGR48a3 by T7 RNA polymerase was injected into defolliculated oocytes. Two to three days later, record- ings were performed in the voltage clamp mode made at the holding potential of -70 mV. A, membrane currents induced by superfusion with 0.1, 0.5, or 4 mM glycine. Strychnine (100 nM) inhibited the response to 0.5 mM glycine by X30% (lower truce, note different scale). Bars indicate duration of agonist and antagonist application. R, agonist dose-response curves obtained with another injected oo- cyte. In different experiments (n = 4), half-maximal responses (EC&J to glycine (0) were seen at 0.75 + 0.25 mM, and to p-alanine (0) at about 7 mM; for taurine (Q-induced responses EC& values could not be calculated.

of the putative transmembrane region M4. To investigate the pharmacological properties of the rat

GlyR (~3 polypeptide, in z&o-synthesized cu3 RNA was in- jected into oocytes. After 2-5 days, the cells were analyzed for glycine, a-alanine, and taurine responses in the voltage clamp mode. Inward currents reversing at the equilibrium potential of chloride (data not shown) were seen with glycine and p- alanine, indicating that functional chloride channels are

PO P20 P40 as-+ ,,r,-_1

+q FIG. 4. Amplification of GlyR subunit transcripts in rat

spinal cord. PCR with ot3, n2*, and otl specific oligonucleotide combinations was performed in parallel using equal amounts of cDNA reversely transcribed from poly(A)+ RNA isolated from rat PO (post- natal day 0), P20, and P40 spinal cord. After agarose gel electropho- resis of the amplification products, specific sequences were visualized with radiolabeled DNA probes of the indicated subunits as detailed under “Materials and Methods.”

formed upon expression of cu3. Membrane currents were ob- served with glycine concentrations between 0.1 and 10 mM, and half-maximal responses obtained at EC& = 0.75 mM (Fig. 3B). This agonist concentration elicits nearly maximal re- sponses with cul subunit receptors generated from the previ- ously characterized rat and human GlyR cDNAs (14, 18, 19). Thus glycine receptors generated from expressed ~y3 sequences display glycine affinities lower than those of al from human and rat and of (~2 from human. Strychnine blocks the opening of channels formed by rat and human cul and human (~2 polypeptides at concentrations of 20-100 nM (14, 18, 19). A similar inhibition of chloride currents was observed here with 100 nM strychnine on oocytes expressing (~3 (Fig. 3A). Thus, a reduced affinity for glycine of the expressed rat a3 receptor can be distinguished from unaltered strychnine sensitivity. The concentrations required to obtain responses with P-ala- nine were in the millimolar range, i.e. similar to those reported for the rat otl subunit, or the human cul and (~2 subunit receptors (14, 18). However, taurine, which elicits large re- sponses on receptors generated from expressed rat and human cul, but not (u2* and (~2 cDNAs, induced only very poor currents in oocytes injected with cu3 RNA (Fig. 3B).

Developmental heterogeneity of GlyR LY subunits has been demonstrated previously (15, 16). To analyze the temporal appearance of (~3 transcripts, we performed PCR amplifica- tion of cDNA synthesized on spinal cord RNA preparations isolated at different stages of development. As shown in Fig. 4, 013 amplification products were barely detectable at birth (PO) but steeply increased at days 20 (P20) and 40 (P40) postnatally. The relative abundance of (~3 transcripts in adult animals resembles the expression of the cul subunit mRNA in postnatal rodents. In contrast, ot2* amplification products were abundant in PO RNA but barely seen at later stages of spinal cord development (Fig. 4). These results indicate that the (~3 sequence encodes a GlyR subunit mainly expressed in adult rats.

In conclusion, the data presented here extend the notion that the GlyR exists in several isoforms in the mammalian central nervous system (14-16). In addition to the previously documented heterogeneity of this receptor in neonatal spinal cord (15-17), GlyR (Y subunit diversity also exists in the adult animal. The precise physiological significance of these mul- tiple (Y subunit variants is presently unclear. However, our expression data suggest that they may serve in assembling GlyR subtypes of different agonist response properties in spinal cord and possibly other regions (12) of the developing and adult brain.

22320 Glycine Receptor a3 Subunit

Acknowledgments-We thank M. Munz for expert technical assist- M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) ante, P. Seeburg for providing the rat brain cDNA library, D. Lan- Nature 328,215-220 gosch for critical reading of the manuscript, and B. Albers for help 12. Grenningloh, G., Pribilla, I., Prior, P., Multhaup, G., Beyreuther,

during its preparation. K., Taleb, O., and Betz, H. (1990) Neuron 4,963-970 13. Grenninzloh. G.. Gundelfineer. E.. Schmitt. B.. Betz. H.. Darli-

1.

2.

3.

4.

5.

6.

7.

8.

9. 10.

11.

REFERENCES

G., Wada, E., Jensen, A., Gardner, P. D., Ballivet, M., Deneris, E. S., McKinnon, D., Heinemann, S., and Patrick, J. (1990) J. Biol. Chem. 265,4472-4482

Boulter, J., O’Shea-Greenfield, A., Duvoisin, R. M., Connolly, J.

Levitan, E. S., Schofield, P. R., Burt, D. R., Rhee, L. M., W&den, W., Kohler, M., Fujita, N., Rodriguez, H. F., Stephenson, A., Darlison, M. G., Barnard, E. A., and Seeburg, P. H. (1988) Nature 335,76-79

son, M G.; Barnard, E. A.: Schofield, P. R., and Seehurg, P. H. (1987) Nature 330, 25-26

14.Grenninzloh. G.. Schmieden. V.. Schofield. P. R.. Seeburz. P. H..

15. 16.

17.

18.

19.

Siddi&e,‘T.,‘Mohandas,‘T. ‘K., Becker, C. M., and Betz, HI (1990) EMBO J. 9, 771-776

Kuhse, J., Schmieden, V., and Betz, H. (1990) Neuron, in press Becker, C.-M., Hoch, W., and Betz, H. (1988) EMBO J. 7, 3717-

3726 Hoch, W., Betz, H., and Becker, C.-M. (1989) Neuron 3, 339-

348 Schmieden. V.. Grennineloh. G.. Schofield. P. R.. and Betz. H.

(1989) EtiBb J. 8, 69&760 Sontheimer, H., Becker, C.-M., Pritchett, D. B., Schofield, P. R.,

Grenningloh, G., Kettenmann, H., Betz, H., and Seeburg, P. H. (1988) Neuron 2,1491-1497

Ymer, S., Schofield, P. R., Draguhn, A., Werner, P., Kohler, M., and Seeburg, P. H. (1989) EMBO J. 8, 1665-1670

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular CloninP:A Laboratorv Manual. Cold Snrine Harbor Laboratorv. Cold Spring Harbor,“NY - -

-

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. N&l. Acad. Sci. U. S. A. 74,5463-5467

Cathala, G., Savouret, J.-F., Mendez, B., West, B. L., Karin, M., Martial. J. A.. and Baxter. J. D. (1983) DNA (N.Y.) 2, 329-

Aprison, M. H., and Daly, E. C. (1978) Adu. Neurochem. 3, 203- 294

Young, A. B., and Snyder, S. H. (1973) Proc. N&l. Acad. Sci. U. S. A. 70, 2832-2836

Young, A. B., and Snyder, S. H. (1974) Mol. Pharmacol. 10,790- 809

20.

21. Pfeiffer, F., Graham, D., and Betz, H. (1982) J. Biol. Chem. 257,

9389-9393 Graham, D., Pfeiffer, F., Simler, R., and Betz, H. (1985) Biochem-

istry 24,990-994 22.

Becker, C.-M., Hermans-Borgmeyer, I., Schmitt, B., and Betz, H. (1986) J. Neurosci. 6, 1358-1364

Betz, H., and Becker, C.-M. (1988) Neurochem. Znt. 13, 137-146 Langosch, D., Thomas, L., and Betz, H. (1988) Proc. N&l. Acad.

Sci. U. S. A. 85, 7394-7398

23.

24. 335

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239,487-491

Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, 25. Kozak, M. (1989) Mol. Cell. Biol. 9, 5073-5080