Neuronal Nicotinic Receptors in the Locust Locusta migratoriaCLONING AND EXPRESSION*

(Received for publication, February 23, 1998, and in revised form, April 28, 1998)

Bernhard Hermsen‡, Eva Stetzer‡, Rudiger Thees‡, Reinhard Heiermann‡, Andre Schrattenholz‡,Ulrich Ebbinghaus§, Axel Kretschmer§, Christoph Methfessel§, Sigrid Reinhardt‡,and Alfred Maelicke‡¶

From the ‡Laboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry,6 Duesbergweg, Johannes-Gutenberg University Medical School, 55099 Mainz and §BAYER AG, Central Research Unit,5090 Leverkusen, Germany

We have identified five cDNA clones that encode nic-otinic acetylcholine receptor (nAChR) subunits ex-pressed in the nervous system of the locust Locusta mi-gratoria. Four of the subunits are ligand-binding asubunits, and the other is a structural b subunit. Theexistence of at least one more nAChR gene, probablyencoding a b subunit, is indicated.

Based on Northern analysis and in situ hybridization,the five subunit genes are expressed. loca1, loca3, andlocb1 are the most abundant subunits and are expressedin similar areas of the head ganglia and retina of theadult locust. Because Loca3 binds a-bungarotoxin withhigh affinity, it may form a homomeric nAChR subtypesuch as the mammalian a7 nAChR. Loca1 and Locb1may then form the predominant heteromeric nAChR inthe locust brain. loca4 is mainly expressed in optic lobeganglionic cells and loca2 in peripherally located so-mata of mushroom body neurons. loca3 mRNA was ad-ditionally detected in cells interspersed in the somato-gastric epithelium of the locust embryo, suggesting thatthis isoform may also be involved in functions otherthan neuronal excitability. Transcription of all nAChRsubunit genes begins approximately 3 days beforehatching and continues throughout adult life.

Electrophysiological recordings from head ganglionicneurons also indicate the existence of more than onefunctionally distinct nAChR subtype. Our results sug-gest the existence of several nAChR subtypes, at leastsome of them heteromeric, in this insect species.

In insects, neuromuscular transmission is mediated by glu-tamate, whereas acetylcholine is the principal neurotransmit-ter in the nervous system (1). A large body of evidence suggeststhe existence of both muscarinic and nicotinic acetylcholine(nAChR)1 receptors in the insect brain, with nAChR-codingRNAs having been identified in several species, including the

fruit fly Drosophila (2), the locust Schistocerca (3), the tobaccohornworm Manduca (4), and the peach-tomato aphid Myzus(5). Considerable pharmacological diversity of nicotinic recep-tors is indicated by the existence of aBTX-sensitive and -insen-sitive receptors (6, 7) and by the rather wide variation ofresponses to nicotinic and non-nicotinic drugs of insect neuronsand membrane preparations (8, 9). In particular, the nAChR ofLocusta migratoria was suggested to have mixed nicotinic andmuscarinic pharmacology (10), which could correlate with thegreater evolutionary age of orthoperians as compared withdipterians.

Vertebrate neuronal nicotinic receptors are quite diverse(11), with to date eight a subunits and three b subunits clonedin the rat. Of these, the a7, a8, and a9 subunits have theunique ability to form functional homomeric receptors (12–14).Various combinations of the other a and b subunits also giverise to functional receptors, as is exemplified by combinationsof a4 and b2 subunits and of a3 and b4 subunits expressed inhippocampal neurons (15). The stoichiometries of heteromericneuronal nAChRs are not yet established.

The homo-oligomeric receptors appear to be the evolutionar-ily oldest (16), which has led to the suggestion that the inver-tebrate neuronal nAChR from L. migratoria, given its broadpharmacology, may be an a7-like homo-oligomeric receptor (17,18). In Drosophila, five different putative nAChR subunitshave been identified, three of which contain the two adjacentcysteines that are characteristic of ligand-binding a subunits(2).

The subunit compositions and stoichiometries of insect nic-otinic receptors are still unknown. This is in part due to the factthat expression in Xenopus oocytes of insect nAChR subunitRNA and cDNA has generally proven to be difficult (19). Forthe same reason, it has not yet been possible to determine theelectrophysiological and pharmacological properties of singlesubtypes of insect nicotinic receptors.

In the present study, we show that in the locust L. migrato-ria at least six different genes exist that encode nAChR sub-units. Four of these genes encode for a subunits. Although wewere unable to demonstrate heterologous functional expressionin Xenopus oocytes of single subunits, or combinations of sub-units, in situ hybridization studies show that the identifiedsubunits are expressed in vivo and probably form functionalreceptors with different quarternary structures. Because insectnAChRs represent important targets for insecticides (20, 21),the structural and functional characterization of such receptorsmay be useful in the context of rational drug design.

EXPERIMENTAL PROCEDURES

Materials—Locust eggs were supplied by Futtertierversand Hintze,Berlin (Germany). Dr. August Dorn (Institute of Zoology, University

* This work was supported by the Stiftung fur Innovation Rheinland/Pfalz, the Fonds der Chemischen Industrie, and BAYER AG, Le-verkusen. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s)AJ000390–000393.

¶ To whom correspondence should be addressed. Tel.: 49 6131395911; Fax: 49 6131 393536; E-mail: alfred.maelicke@uni-mainz.de.

1 The abbreviations used are: nAChR, nicotinic acetylcholine recep-tor; ACh, acetylcholine; aBTX, a-bungarotoxin; kBTX, k-bungarotoxin;dNTP, desoxynucleotide triphosphate; TM, transmembrane domain;dn, embryonic day.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 29, Issue of July 17, pp. 18394–18404, 1998© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Mainz) provided live adult locusts, and Dr. Heinz Breer (Institute ofZoology, Stuttgart-Hohenheim; Germany) provided muscle and gangliatissue from adult locusts.

Preparation of a Locust-specific Genomic nAChR Probe—A L. migra-toria genomic library was constructed using the lambdoid phageNM1149 in combination with the hfl mutant Escherichia coli strainPOP13b, which suppresses the lytic cycle of non-recombinant phages.Chromosomal DNA was prepared from muscle tissue of adult locusts asdescribed (22). DNA was restricted with EcoRI, fractionated on a 0.7%agarose gel, and fragments from 3.8 to 5.4 kilobase pairs length wereisolated. Samples of 100 ng of fractionated DNA were ligated with 1 mgof EcoRI-restricted NM1149-DNA and packaged. Packaging efficiencywas 8 3 105 lytic plaques per 100 ng of Locusta DNA.

The E. coli strain POP13b was infected with 1.5 3 106 recombinantplaques, and the genomic library was screened using a [a-32P]dATPrandomly labeled 650-bp fragment from the Drosophila ARD cDNA(23). Filters were hybridized overnight and washed with low stringency(23 SSC, 55 °C; 203 SSC, saline sodium citrate: 0.3 M sodium citrate,3 M NaCl). After 48 h exposure two positive signals were identified.These phages were plated with lower density and hybridized. Tworecombinants were isolated and characterized. A 177-bp AluI fragmentof one of these recombinants coded for the full second transmembranedomain (TM2), the following short loop, and part of the third trans-membrane domain (TM3) of a nAChR. This fragment was used asscreening probe in further experiments.

Isolation of cDNA Clones—A L. migratoria cDNA library was con-structed in lgt11. Poly(A)1 RNA was extracted from ganglionic tissueand reverse-transcribed to double strand DNA using hexanucleotideprimers. cDNA was ligated to EcoRI linkers and cloned into lgt11. E.coli strain BHB2600 was infected 5.5 3 106 recombinants. 600,000phages were plated with high density (50,000 plaques/13-cm dish).Screening was performed using randomly primed [a-32P]dATP-labeled177-bp AluI fragment. Filters were washed under low stringency (23SSC, 55 °C). Positive phages were re-plated with lower density (100–200 plaques/8-cm dish) and re-hybridized.

Cloning of the First 200 Amino Acids of the Loca1 cDNA Clone—RNAwas prepared from d9 embryos, and single strand cDNA was preparedfrom 5 mg of total RNA using a mixture of oligo(dT)12–18 and randomprimers. Reaction was carried out using 400 units of Superscript Re-verse Transcriptase (Life Technologies, Inc.) following the provider’sprotocol.

1 ng of single strand cDNA was amplified in a first polymerase chainreaction (PCR) using 39-primers highly specific for Loca1 (59-CTC-GAGGGACATGTAGAACTCGGAGAGGTC-39) and a mix of two 59-primers (59-ACCGCCTCATCAGGCCTGTCACCAACAACTCCGA-39and 59-ACCGCCTCATCAGGCCTGTCGGCAACAACTCGGA-39) de-signed to highly homologous regions of loca2 and loca3 cDNA clones (aa15–21 of loca2 and aa 4–10 of loca3, respectively). Both 59-primerscontained a silent mutation to introduce a StuI site (bold letters) whichwas later used to fuse the rat a3 nAChR signal peptide to the locust a1clone. PCR products were diluted 1:100 and re-amplified in a secondPCR using the same 59-primers and another 39-primer (59-GGCAG-GCACCTCGAGGATGTCCCA-39) designed to the already amplifiedfragments. Both PCRs were carried out in a volume of 20 ml under thefollowing conditions: 1 3 Taq buffer (Life Technologies, Inc.), 10 pM ofeach primer, 5 mM MgCl2, 200 mM dNTP, 50 mM Tris-HCl. pH 9.5, 2.5units of Taq Polymerase (Life Technologies, Inc.). After incubation at94 °C, 5 min amplification was carried out in a Perkin-Elmer Thermo-cycler Gene AmpTM PCR System 2400, running 39 cycles (94 °C, 1 min;58 °C, 1, 5 min; 72 °C, 2, 5 min), and finally incubating at 72 °C, 10 min.PCR products were fractionated, and 600-bp fragments were cloned intopSL1180 vector (Amersham Pharmacia Biotech). Clones containingloca1-specific sequences were isolated by colony hybridization (Sam-brook et al., 1989) using a [a-32P]dATP-labeled oligo (59-CTCGAGGGA-CATGTAGAACTCGGAGAGGTC-39), which is highly specific for loca1.They were then sequenced, and one of them was fused to the partialloca1 cDNA clone.

Construction of Full-length cDNAs—The BamHI/EcoRI fragment ofthe rat a3 nAChR cDNA (24) and the locust a2, a3, and b cDNAs werecloned into pBluescript® KS1 (Stratagene). A StuI site was introducedinto the 59-end of these clones by PCR without changing the Arg/Proamino acid sequence at this site. PCR was performed with 1 ng of eachclone under the same conditions as described above. Amplification ofthe locust clones was carried out using a 39-primer, designed to thepBluescript® KS1 sequence (59-AACAGCTATGACCATG-39), and thefollowing specific 59-primers containing a silent mutation (in order tointroduce a StuI site; see in bold letters) (loca2, 59-ACCGCCTCATCAG-GCCTGTCACCAACAACTCCGA-39; loca3, 59-ACCGCCTCATCAGGC-CTGTCGGCAACAACTCGGA-39; locb, 59-ACAAGCTCATCAGGCCT-GTGCAGAACATGACGCA-39). Amplification of the rat a3 nAChRfragment was carried out with 2 ng of cloned DNA using the followingprimers: 59-GTAAAACGACGGCCAGT-39, 59-CCTCCGAATTCTC-

FIG. 1. Current responses of a neu-ron from the locust head ganglion toapplications of acetylcholine at 270mV membrane potential. The dose-re-sponse curve was fitted by the Hill equa-tion. All currents were normalized to themean amplitude elicited by 10 mM AChbefore and after each test concentrationwas applied. The EC50 for ACh was 17.9 63.4 mM (mean 6 S.D., n 5 7 cells). Theupper inset shows typical ACh responsesat different concentrations.

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CGGA-GATGATCTCGTTGTAAT-39). PCR products were cloned intopBluescript® KS1 and sequenced. The StuI site was then used to fusethe rat a3 nAChR signal sequence to each of the locust cDNA clones.

In Situ Hybridization—In situ hybridization was performed withdigoxigenin-labeled RNA probes. RNA probes were randomly labeledwith digoxigenin according to the protocol provided by the manufac-turer (Boehringer Mannheim, Germany). In order to obtain isoform-specific probes, sequence regions with lowest homology were selected,i.e. for loca1 and loca2 clones from the “cytoplasmic loop” between TM3and TM4, for locb from parts of the 59-translated region, for loca3 fromthe 39-untranslated region, and for loca4 from the 59-untranslatedregion. The isoform specificity of the RNA probes was confirmed bySouthern analysis.

Whole embryos and head ganglia with attached optic lobes andretina from adult locusts were dissected out of the eggshell and indi-vidual adult insect, respectively, placed in embedding medium (TissueTec, Miles), and shock-frozen with dry ice. They were kept at 270 °Cuntil use. 6–9-mm frozen sections were obtained at 220 °C using a Slee(Mainz, Germany) cryostat.

Cryosections were fixed with paraformaldehyde (4%) for 10 min,washed 4 times for 5 min with PBS supplemented with 0.1% Tween 20(PBS-T), and incubated in hybridization buffer (50% formamide, 53SSC, 50 mg/ml tRNA, 50 mg/ml heparin, 0.1% Tween 20) for 1 h at 50 °C.Digoxigenin-labeled RNA probes were added, and the cryosections wereincubated overnight. After five washes for 20 min in 23 SSC, 50%formamide, and treatment with RNase A (25 mg/ml) and RNase T1 in23 SSC, 8 further washes were performed in which the SSC buffer wasdiluted out with PBS-T in a stepwise fashion. After incubation for 1 hwith PBS, supplemented with 2 mg/ml bovine serum albumin and 0.1%Triton X-100, the sections were incubated for 30 min with an anti-digoxigenin antibody conjugated with alkaline phosphatase (Boeh-ringer Mannheim, Germany). After two washes with 100 mM Tris-HCl,150 mM NaCl, pH 7.5, the sections were incubated with 100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5, supplemented with the dyereagents nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phos-

phate. After washing the sections for 5 min in PBS, they were coveredin PBS, 50% glycerol and were analyzed under the microscope.

Southern and Northern Blot Analysis—10 mg of chromosomal DNAprepared from muscle tissue of adult locusts was completely restrictedwith EcoRI, BamHI, and HindIII, fractionated, and blotted onto a nylonmembrane. The genomic blot was hybridized with the randomly primed[a-32P]dATP-labeled AluI fragment and was washed under moderatestringency (65 °C; 13 SSC).

RNA was prepared from embryos d4–d9 after egg laying. 1 mg ofRNA was used to prepare poly(A)1 RNA. Every 5 mg of poly(A)1 RNA ofall six developmental stages was submitted to electrophoresis and blot-ted onto a nylon membrane. Blots were hybridized with three randomprimed [a-32P]dATP-labeled probes: a 420-bp HinfI/HinfI fragment ofthe loca2 cDNA, a 230-bp EcoRI/SphI fragment of the locb1 cDNA, anda 370-bp EcoRI/SphI fragment of the loca1 cDNA. Blots were washedwith 13 SSC, 65 °C.

Fragments of nAChR a Subunits Obtained by Expression in E. coliand Binding Studies with a-Bungarotoxin—cDNA clones were obtainedfrom the following sources: Drosophila a1 from Marc Ballivet, Geneva,Switzerland (25); Drosophila a2 from Eckart Gundelfinger, Magdeburg,Germany (26); and Torpedo a from Toni Claudio, New Haven, CT.cDNA fragments of the N-terminal extracellular regions were ex-pressed either as maltose-binding protein fusions using pMAL-c2 vectorfrom New England Biolabs (Schwalbach, Germany) or as glutathioneS-transferase fusions using pGEX-4T-1 vector (Amersham PharmaciaBiotech). Fusion proteins were prepared from isopropyl-1-thio-b-D-ga-lactopyranoside-induced E. coli transformants (E. coli strain DH5a,Life Technologies, Inc.) of flask submersed cultures. E. coli pellets weresonicated in lysis buffer, and after centrifugation, the supernatant wasdiluted and applied to affinity chromatography. Eluted nAChR fusionproteins were dialyzed, and the purity was determined by capillaryelectrophoresis and SDS-PAGE. Based on these methods, the purity offusions proteins was generally better than 85%. In selected examples(Torpedo a fragment), the fusion protein was proteolytically cleaved(factor Xa), and the nAChR fragment was isolated, and terminal pep-

FIG. 2. Current responses of twoganglion cells to ACh and nicotine,and dose-response curves for nico-tine. Upper, current responses of two dif-ferent locust ganglion cells to ACh andnicotine at 270 mV membrane potential.Note the very different response ampli-tudes to nicotine. Lower, dose-responsecurves for nicotine taken from the sametwo cells. The current values were nor-malized to the maximal current elicitedby a saturating concentration of ACh oneach cell.

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tide sequencing and mass spectrometry were performed. These dataconfirmed the primary structure of the nAChR fragments. Fusion pro-teins were separated on a, SDS-polyacrylamide gel, which was thenblotted onto a nitrocellulose membrane. The membrane was incubatedwith 1029 M 125I-aBTX at 4 °C, 30 min, and washing was performed 5 3for 30 min with 13 PBS. 125I-aBTX was obtained from AmershamPharmacia Biotech (Braunschweig, Germany).

Preparation of Locust Neurons and Electrophysiological Record-ings—Head (supra-esophageal) ganglia and optic lobes from individualadult L. migratoria were dissected out and placed into dissociationsolution (Sigma-Aldrich, Deisenhofen, Germany). Dispase (2 mg/ml,Life Technologies, Inc., Eggenstein, Germany) was added and incu-bated for 5 min at 37 °C. The material was then centrifuged, and thepellet was resuspended in culture buffer and was dissociated by gentleaspiration with a fire-polished Pasteur pipette (27). Cells were platedonto glass coverslips that were pre-coated with concanavalin A (400mg/ml, Sigma) and laminin (4 mg/ml, Sigma). The cultures were kept atroom temperature and used for electrophysiological measurements onthe following 2 days.

For electrophysiological recordings, the whole cell patch clamp tech-nique was used. Microelectrodes were pulled from borosilicate glasscapillaries (Hilgenberg, Malsfeld, Germany). The resistance of the fire-polished pipettes was 4–7 megohms, using the internal and externalsolutions described below. All experiments were performed at roomtemperature (22–25 °C).

Cells were placed in a perfusion chamber at approximately 0.5-mlvolume and superfused continuously (flow rate 3 ml/min) with externalbath solution. The bath solution contained (in mM): 150 NaCl, 4 KCl, 2MgCl2, 2 CaCl2, 10 HEPES, pH 7.4, adjusted with NaOH. The (intra-cellular) pipette solution contained (in mM): 120 CsF, 30 CsCl, 10Cs-EGTA, 1 CaCl2, pH 7.4 adjusted with CsOH.

Currents were measured using an L/M-EPC-7 patch clamp amplifier(List, Darmstadt, Germany). The holding potential was 270 mV. Cur-rent records were low pass Bessel filtered at 315 Hz and digitized at

1-kHz sample rate. Data storage and analysis were performed with thepClamp version 6.03 software package (Axon Instruments, Foster City,CA). Test substances were applied to the cells using the U-tube reversedflow technique (28) with applications of 1–2 s duration at intervals of 1min. Acetylcholine chloride, cytisine, and coniine were obtained fromSigma. Nicotine bitartrate was obtained from RBI (Natick, MA). Drugswere stored frozen as stock solutions (10 or 100 mM in water) andthawed and diluted on the day of the experiment.

RESULTS

Electrophysiological Studies Suggest Several Subtypes ofNeuronal nAChR in the Locust L. migratoria—The dissociatedneurons obtained from head (supra-esophageal) ganglia andoptic lobes of L. migratoria and cultured for 1–2 days on glasscoverslips were large, round cells between 30 and 120 mm indiameter, frequently having short protrusions. These cells in-variably responded to test applications of 10 mM acetylcholine(ACh) with a fast inward current of between 100 and 2000 pAat 270 mV clamp potential. Dose-response curves obtainedwith ACh on head ganglion cells (Fig. 1) yielded an EC50 valueof 17.9 6 3.4 mM (n 5 7), whereas an EC50 of 19.1 6 8.9 (n 5 4)was measured for optic lobe cells (not shown). In both cases,other nicotinic agonists such as (2)-nicotine or cytisine elicitedresponses that, even at saturating concentrations, remainedwell below the maximal currents induced by ACh.

The time course of the response to ACh was quite variablefrom one cell to another. In addition to the rapidly desensitiz-ing component present in most cells, some cells also exhibiteda non-desensitizing response to ACh, suggesting the existenceof (at least) two nAChR subtypes. Further evidence was pro-vided by dose-response curves for nicotine on cells in which oneor the other type of ACh response predominated (Fig. 2). In acell with mostly rapidly desensitizing ACh-induced currents,nicotine was a weak partial agonist with EC50 .20 mM (29). Bycontrast, another cell with non-desensitizing ACh currents re-sponded sensitively to nicotine, with an EC50 of ,1 mM. Sincemost cells contained both subtypes of nAChR in varying pro-portions, the effective EC50 values for nicotine were quite vari-able (e.g. 9.4 6 11.4 mM, n 5 5 in optic lobe cells). Similarresults were obtained with cytisine, whereas coniine acted asagonist exclusively at the non-desensitizing nAChR subtype.Taken together, these data confirm the existence of functionalnAChR ion channels in locust neurons and clearly suggest thatthere exist at least two pharmacologically distinct subtypes ofnAChR in the cells studied.

The L. migratoria Genome Contains Several Genes Encodingfor nAChR Subunits—A genomic library was prepared frommuscle tissue of adult L. migratoria nAChR and was screenedwith a 32P randomly labeled 650-bp long fragment from Dro-sophila ARD2 cDNA (23) encoding the transmembrane regionsTM1–TM3. We obtained among other clones a 4.1-kilobase pairlong genomic fragment that displayed 69.2% homology to theDrosophila ARD2 sequence, 81.6% to the Drosophila ALS se-quence, and 75.9% to the chick a4 sequence. An AluI fragment(177 bp) of the genomic clone, containing mostly coding se-quences (140 bp) and encoding the full second transmembrane

FIG. 3. Southern blot analysis of L. migratoria chromosomalDNA treated with three different restriction endonucleases. L.migratoria chromosomal DNA was obtained from muscle tissue. 10 mgof DNA were treated with the restriction enzymes EcoRI (E), HindIII(H), and BamHI (B), respectively, fractionated, and transferred onto anylon membrane. The blot was hybridized under low stringency condi-tions with a 32P-labeled genomic probe of 177 bp, containing mostlycoding sequences (140 bp) and encoding the full second transmembranedomain (TM2), the following short loop, and part of the third trans-membrane domain (TM3). The restriction fragment lengths were(EcoRI): 20.0, 15.0, 9.0, 8.1, 8.0, 4.7, 4.1, and 3.8; (HindIII): 15.0, 8.9,8.1, 7.2, 6.4, 5.7, and 5.0; (BamHI): 22.0, 20.0, 16.0, 12.0, 9.1, and 7.0.

FIG. 4. Hydropathy profile of Loca2subunit and generalized domainstructures of L. migratoria nAChRsubunits. The amino acid residues arenumbered according to the Loca2 se-quence (Fig. 4). M1–M4 refer to putativetransmembrane domains.

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domain (TM2), the following short loop, and part of the thirdtransmembrane domain (TM3), was prepared, randomly la-beled with [32P]dATP, and used as probe in a genomic blot ofpurified DNA from muscle tissue of adult locusts that wascleaved with the restriction endonucleases EcoRI, HindIII, andBamHI. As shown in Fig. 3, at least six bands were detected ineach lane, suggesting that this number represents the approx-imate number of nAChR-coding genes that exist in L.migratoria.

Isolation and Sequencing of Five cDNAs Coding for nAChRSubunits—After initial information was obtained by Northernanalysis (see later for details) for the developmental stages atwhich nAChR subunit mRNA is expressed in the locust, weprepared a cDNA library from late embryonic stage. By usingthe 32P-labeled genomic AluI fragment described above, wescreened the cDNA library at relatively high stringency. Weobtained several cDNA clones which coded for four a subunitsand one b subunit. The cDNA clones loca2, loca3, and locb1encoded for mature subunits, whereas clone loca1 missed thenucleotides encoding for the first approximately 200 aminoacids of the mature protein. All four cDNA clones missed the59-terminal sequences encoding the signal sequences. AnothercDNA clone isolated encoded the full N-terminal sequence of ana subunit (loca4) but extended only to the end of transmem-brane domain 2. The complete sequence of loca1 was obtainedby PCR. Attempts to also obtain by PCR the signal sequences ofthe four full-length cDNA clones were not successful. The ma-ture proteins of L. migratoria Loca1-a3 subunits consist of 559,515, and 540 amino acids, respectively, with predicted molec-ular masses of 61.5, 56.7, and 59.4 kDa. The Locb subunitconsists of 497 amino acids with a molecular mass of 54.7 kDa.The nucleotide sequences of the four full-length clones havebeen reported to the EMBO nucleotide sequence Data Bank(accession numbers AJ000390–000393). Since none of the fivecDNAs was identical in sequence to the exon sequence of thegenomic AluI fragment used as screening probe (homologies

are 79–89% for the a clones, 64% for the b clone), there mustexist at least one more gene in L. migratoria that encodesnAChR subunits.

Properties of Sequences and Homology to Other InsectnAChRs—In Fig. 4 (upper panel), the hydropathy plot of theLoca2 isoform is representatively shown. As is typical of allisoforms of nAChR identified, it displays the pattern of fourputative transmembrane domains (TM1–4) that is common forthe superfamily of ligand-gated ion channels. The sequencescoding for the a-isoforms also contain in their N-terminal ex-tracellular domain the four conserved cysteines (Fig. 4, lowerpanel), including the two vicinal ones just in front of the firsttransmembrane domain, and two putative N-glycosylationsites. The cytoplasmic domain between TM3 and TM4 is quitevariable in size (199, 147, 185, and 156 amino acids in Loca1–3and Locb1, respectively) and contains several putative phos-phorylation sites. In the Locb1 sequence, this region contains aputative phosphorylation site for cAMP-dependent kinase, aswas also found in non-a subunits from Torpedo (30) and Dro-sophila (23).

Sequence homologies of the four full-length clones of the L.

FIG. 5. Phylogenetic tree of insect nAChR isoforms. The phylo-genetic tree (top) was constructed on the data presented in the table(bottom). Amino acid identity between complete amino acid sequencesexcept the cytoplasmic loop was obtained using Clustal method withPAM250 weight table. Accession numbers are as follows: ALS, P09478;Da2/SAD, P17644; ARD, X04016; SBD, X55676; Mpa1, X81887; Mpa2,X81888; aL1, P23414; MARA1, Y09795. NAChR isoforms with 80%, ormore, amino acid sequence identity to particular Loc subunits areindicated in bold in the table. These subfamilies each represent aseparate evolutionary branch.

FIG. 6. Binding of a-bungarotoxin to fusion proteins contain-ing fragments of the N-terminal extracellular region of nAChR asubunits from several species. Samples of affinity purified fusionproteins containing nAChR fragments from Torpedo a (aa 1–246, lane1), Drosophila a2 (aa 3–226, lane 2), Loc a3 (aa 126–229, lane 3), Loc a3(aa 12–227, lane 4), Loc a2 (aa 1–222, lane 5), and Drosophila a1 (aa83–223, lane 6) were separated on SDS-PAGE and stained with Coo-massie (upper) or blotted onto nitrocellulose for Western blotting using1029 M mono-iodinated 125I-aBTX (bottom). The complex pattern ofprotein bands in lanes 4 and 5 is probably caused by premature trans-lation stop of malE-nAChR fusion polypeptides. Binding of aBTX wasdetected for Torpedo-a, Loca3, and Da1.

FIG. 7. Developmental stage-specific expression of L. migrato-ria nAChR subunits a1, a2, and b1 as studied by Northern blotanalysis. 5 mg of poly(A)1 RNA of each embryonic day (d4–9) weresubmitted to electrophoresis and blotted onto a nylon membrane. Hy-bridization was performed with randomly 32P-labeled cDNA fragmentsof clones loca1, loca2, and locb1. A, 420-bp long HinfI/HinfI fragmentfrom loca2; B, 370-bp long EcoRI/SphI fragment from loca1; C, 230-bplong EcoRI/SphI fragment from locb1. Size markers were mouse rRNA(28 S and 18 S). All three blots show beginning subunit mRNA expres-sion at day 6 after egg laying, with maximal expression on d8.

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migratoria nAChR subunits to those of other species are re-ported in Fig. 5, top and bottom.

Attempts to Express the L. migratoria nAChR cDNAs andmRNA in Xenopus Oocytes—Functional expression in Xenopusoocytes of insect nAChR following injection of mRNA into thecells has been reported for the cloned a subunit from the locustSchistocerca gregaria (3). Functional channels were formedthat were gated by micromolar concentrations of nicotine andthat were blocked by aBTX, kBTX, strychnine, and bicuculline.These results suggest that in Schistocerca there exists a homo-meric nAChR that has similar physiological properties as the

nicotinic responses recorded from insect neurons. In contrast,Drosophila nAChR a subunits so far could only be co-expressedin Xenopus oocytes with the chick neuronal b2 subunit (31).

We have undertaken various attempts, so far in vain, toexpress the cloned L. migratoria nAChR subunits in Xenopusoocytes. To achieve expression, we introduced by silent mu-tagenesis a StuI restriction site at the 59-end of each clone, andby using this site, we attached rat a3 signal sequence to theLocusta cDNA clones. We then injected into Xenopus oocytessingle subunit cDNA and cDNA mixtures of different subunitsor in vitro transcribed mRNA, neither treatment led to any

FIG. 8. Expression of nAChR isoforms in head ganglia of the adult locust. A, schematic representation (modified after Ref. 51) of theanatomic organization of the adult locust head ganglia with the supra-esophageal ganglion (mushroom body, framed) which consists of ganglioniccell bodies (gcb) and neuropil (np), the optic lobes, including the lamina ganglionaris (lg), medulla externa (me), medulla interna (mi), and the opticlobe connective (ole), and the retina (r). The area from which the micrographs B–F were taken is framed. B–F, in situ hybridizations of mRNAencoding for a1 (B), a2 (C), a3 (D), a4 (E), and b1 (F) nAChR isoforms using cryostat sections of isolated locust brains. Digoxigenin-labeledantisense-RNA probes were used (for details, see “Experimental Procedures”). p, perineurium. Bars, 40 mm. In the areas depicted, mRNA codingfor the subunits a1, a2, a3, and b1 was detected in the ganglionic cell bodies (see also inset of C) but not in the neuropil (the approximate bordersbetween ganglionic cell bodies and neuropil are indicated by dashed lines).

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FIG. 9. Expression of nAChR isoforms in the retina and optic lobes of the adult locust. A and B, schematic representations of the retinaand the accessory optic lobe, and the cellular component of an ommatidium (modified after Ref. 51). The areas depicted in the in situ hybridizationsof C–G are framed. C–G, in situ hybridizations with digoxigenin-labeled RNA probes for a1 (C), a3 (D), a4 (F and G), and b1 (E) nAChR isoforms,of cryostat sections of the peripheral part of the retina (C–F), and of the lamina ganglionaris and neighboring ganglionic cell bodies (G). Theabbreviations used are: c, cornea; cc, crystal cone; gcb, ganglionic cell bodies; lg, lamina ganglionaris; me, medulla externa; mi,

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significant channel activity. We also were unable to reproducefor any of the loca clones the successful co-expression in Xeno-pus oocytes of Drosophila a subunit with the vertebrate b2clone (31). The inability of the cloned Locusta subunits to formfunctional channels in Xenopus oocytes may be due to inappro-priate assembly of insect receptors in this ectopic expressionsystem (2, 19), or to missing or inappropriate post-translationalmodifications (32, 33), or to additional as yet unidentified sub-units that are required for assembly and/or channel function.That functional ion channels are formed from L. migratorianAChR subunits was shown by Hanke and colleagues (18, 34)who reported electrophysiological recordings from affinity pu-rified and reconstituted nAChR protein.

The Loca3 nAChR Isoform Binds a-Bungarotoxin—Bindingof the snake neurotoxin aBTX to L. migratoria nAChR isoformswas studied by Western blotting. For this purpose, fusion pro-teins between either maltose-binding protein or glutathioneS-transferase and fragments of nAChR a subunits were ex-pressed in E. coli, and the purified fusion proteins were appliedto Western blotting using as probe 125I-labeled aBTX. As isrepresentatively shown in Fig. 6, the Loca3 fusions (containingaa 126–229 and aa 12–227, respectively) and (as controls) theTorpedo a fusion (aa 1–246) and Drosophila a1 (ALS) fusion (aa83–223) bound aBTX, whereas the Loca2 fusion (aa 1–222) andthe Drosophila a2 fusion (aa 3–226) did not bind 125I-aBTX. Toexclude the possibility of nonspecific binding of aBTX to thefusion partner, nAChR fragments were released by proteolyticcleavage from fusion proteins and were then tested in Westernblots for toxin binding. These experiments (not shown) con-firmed the above findings. In other Western blotting experi-ments (not shown), binding of aBTX to Loca3 (aa 12–290),Torpedo a1 (aa 143–201), rat a1 (aa 1–210), and rat a7 (aa80–213) was demonstrated. Selective binding of aBTX byLoca3, as compared with Loca2, cannot be explained by differ-ences in sequence in the region around the two adjacent cys-teines of a subunits but rather appears to be due to attachmentpoints for the toxin that are located in other sequence region(s).That the binding site for aBTX is discontinuously distributedwithin the N-terminal region of the a subunit has previouslybeen reported for nAChRs from other species (34).

From its aBTX binding properties, Loca3 is a candidate for ahomo-oligomeric nAChR, such as the a7 subtype of mammals(11, 35). As reported above though, we have been unable so farto functionally express this or other isoforms in Xenopus oo-cytes, as was achieved for other homo-oligomeric nAChR (13,36). We therefore do not know whether the Loca3 isoformindeed forms a functional channel with the typical properties ofhomo-oligomeric nAChRs, e.g. fast desensitization and sensi-tivity to epibatidine and choline (37).

Western blotting with the same fusions proteins was alsoperformed with the antibody WF6 which competes with AChand competitive agonists and antagonists (including aBTX) forbinding to the Torpedo nAChR (34, 38, 39). Selective binding ofWF6 was observed only to the Loca3 fusion proteins and (ascontrols) to those from Torpedo a and Da1 (data not shown).These observations agree with previous findings in that theattachment point patterns within the binding sites for aBTXand WF6 seem to be overlapping, albeit distinct (40).

Temporal and Spatial Expression of L. migratoria nAChRmRNA in the Developing and Adult Insect—Initial informationon developmental stage-specific expression of L. migratoria

mRNA was obtained by Northern analysis (Fig. 7). We em-ployed randomly labeled 32P-cDNA probes that were selectedon the basis of minimal sequence homology between each otherand that did not cross-react with the other cDNA clones, as wastested by Southern analysis (not shown). As is representativelyshown for loca1, loca2, and locb1 in Fig. 7, the nAChR subunitRNAs begin to be expressed in late embryonic development, i.e.around d7 after egg laying and 3 days before hatching of thesehemi-metabolic insects. Expression increased and reached amaximal level approximately 1 day before hatching. Northernblots probed with the b cDNA probe always showed two tran-scripts (6.1 and 4.1 kilobase pairs), suggesting cross-hybridiza-tion with a second (as yet unidentified) b subunit mRNA.

Temporal and spatial expression of L. migratoria nAChRmRNA was studied in further detail by in situ hybridizationwith subunit-specific RNA probes of frozen sections of 6–9 mmthickness. In the scheme of Fig. 8A, the areas are indicated inwhich nAChR mRNA was detected in the adult locust; theseare the two-paired head ganglia, called mushroom bodies, thepaired optic lobes, and the retina. The mushroom bodies arecomposed of large neurons, the cell bodies of which are locatedperipherally, whereas the neurites form the central neuropil.The cell bodies of the optic lobe neurons are clustered in astructure that is close to the retina (see also Fig. 9A).

As shown in Fig. 8, B–F, mRNA encoding loca1-3 and locb1,but not loca4, was detected in the mushroom bodies of the adultlocust. The strongest expression was observed for loca1 mRNA,in the cytosol of neurons whose cell bodies are located in thecenter of the mushroom bodies. In roughly the same area, loca3and locb1 mRNA was detected. loca3 mRNA was expressed toa much lower extent than loca1 or locb1. loca4 mRNA was notdetected in mushroom bodies. loca2 mRNA was more abundantin peripherally located cell somata. Specificity of the hybridiza-tions is indicated by the cellular staining pattern which wasconfined to the cytosol and did not include the cell nuclei (seeinset of Fig. 8C). As a general observation, the neurons con-taining Locusta nAChR mRNA were intermingled with unla-beled nerve cells, suggesting that not all neurons of the locustexpress nicotinic receptors. The similar expression patterns ofloca1, loca3, and locb1 suggest that these nAChR subunits mayform a single (consisting of all three subunits) or two separate(a homo-oligomeric and a hetero-oligomeric one) receptorsubtypes.

Expression of nAChR isoforms in the retina and optic lobes isshown in Fig. 9. In the retina, loca1 mRNA was most abundant,followed by locb1 and loca3 mRNA. loca2 and loca4 mRNAwere absent. Transcripts of the three subunits were located inprimary and secondary pigment cells which surround the crys-tal cones. Whereas loca4 mRNA was absent in the retina (Fig.9F), it was detected in the ganglionic cell bodies of the laminaganglionaris of the optic lobes where it was located in cellbodies that are situated close to the fibrous part of the opticlobe (Fig. 9G). As concluded above from the expression patternsin the mushroom bodies, Loca1, Loca3 and Locb1 may form asingle or two separate nAChR subtypes.

As demonstrated in Fig. 10, the expression patterns ofnAChR isoforms in the d8 locust embryo are quite differentfrom those in the adult locust. Whereas locb1 mRNA wasabundantly expressed in the protocerebral lobe, the developingmushroom body (Fig. 10F), the a subunits were not expressedin this area, to any comparable extent. This suggests that the

medulla interna; o, ommatidium; pc, photoreceptor cell; ppc, primary pigment cell; spc, secondary pigment cell. Bars, 20 mm. In the areas depicted,mRNA coding for the subunits a1, a3, and b1 was detected in primary and secondary pigment cells of single ommatidia in the retina. In contrast,a4 mRNA was expressed in ganglionic cells of the optic lobe. Dashed lines in G indicate the approximate borders between ganglionic cell bodiesand the lamina ganglionaris. Inset, a4 mRNA was located in the perinuclear region of ganglionic cell bodies.

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FIG. 10. Expression of nAChR isoforms in the brain and periphery of a d8 embryo. A, overview of an in situ hybridization with ab1-specific RNA probe of a cryostat section through a d8 locust embryo; the areas depicted in the in situ hybridizations of B–H are framed. B–H,in situ hybridizations with digoxigenin-labeled RNA probes for the a1 (B), a2 (C), a3 (D and G), a4 (E), and b1 (F and H) nAChR isoforms, ofcryostat sections of the embryonic head ganglion and the ectodermal eye anlage (B–F), and the somatogastric part of the locust embryo (G and H).The abbreviations used are: np, neuropil; pl, protocerebral lobe; e, ectoderm; ola, optic lobe anlage; se, somatogastric epithelium; gcb, ganglionic

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developing mushroom bodies of embryonic day 8 do not yetexpress functional nAChR and that pre-expression of a struc-tural subunit may be required for functional assembly of het-eromeric locust nAChRs. locb1 mRNA was also detected in theectoderm anlage from which the eye develops (Fig. 10F).

Outside of the head area, loca3 mRNA was mainly detectedin isolated cells interspersed in the somatogastric epithelium(Fig. 10G). Additional limited expression of loca3 mRNA wasobserved in peripheral ganglion cells distributed in the mesen-chyme below the somatogastric epithelium. In contrast, locb1mRNA was exclusively found in peripheral ganglionic cells(Fig. 10H).

DISCUSSION

At Least Six nAChR Subunits Are Expressed in the Locust—They form several functionally distinct subtypes. In the courseof this study, we have identified in the locust L. migratoria sixnAChR subunit genes. Three cDNAs encoding a subunits andone cDNA encoding a b subunit were obtained as full-lengthclones. In addition we have isolated a partial nAChR a subunitcDNA and have identified in Northern blots (Fig. 3) a sixthmRNA which probably encodes another b subunit. This is thelargest number of nAChR subunit genes so far identified in aninvertebrate species, and our results clearly contradict previ-ous suggestions of a single homomeric nAChR in L. migratoria(17). In SDS-PAGE of Torpedo nAChR, the four subunits arebetter separated than expected from their differences in molec-ular mass (between 50.2 and 57.6 kDa) (50). In contrast, the gelpattern of the affinity purified Locusta nAChR protein sug-gested a single polypeptide of approximately 65 kDa (17,whereas the molecular weights calculated from the amino acidsequences vary by approximately the same amount (between54.7 and 61.5 kDa) as the Torpedo subunits. These differencesin SDS-PAGE resolution are probably due to variations in thelevels of posttranslational modifications.

Our cloning data clearly suggest that several subtypes ofnicotinic receptors exist in the locust. These findings are sup-ported by electrophysiological recordings from head ganglianeurons (Figs. 1 and 2) which indicate at least two pharmaco-logically distinct nAChR subtypes (9).

Based on Northern blot and in situ hybridization studies(Figs. 7–10), all six of the identified nAChR subunit genes areexpressed. From the local positions and sizes of cells in themushroom body area in which nAChR-mRNA was detected,expression is probably confined to neurons. This is in contrastto an immunohistochemical study of nAChR expression in thelocust Schistocerca gregaria (41) which suggested expressionalso in glia cells. The general staining pattern with the anti-body used in that study is consistent with the present study inthat all principal neuropils were positive.

The spatial expression patterns of the Locusta nAChRmRNAs are consistent with the existence of several receptorsubtypes. Loca1, Loca3, and Locb1 are the isoforms most abun-dantly expressed in the head ganglia and the retina, whereasLoca4 is mainly expressed in optic lobe ganglionic cells. Basedon its aBTX binding properties (Fig. 6), Loca3 is a candidate fora homomeric nAChR. Extending this line of arguments, Loca1and Locb1 may form the predominant hetero-oligomericnAChR of the locust. In the absence of functional expressionstudies in Xenopus oocytes or other ectopic expression systems,it cannot be excluded though that Loca1, Loca3, and Locb1together form a heteromeric nAChR (42–45). The early pres-

ence of Locb1 in the embryo of the locust suggests that itsexpression may be a prerequisite for functional assembly ofsome hetero-oligomeric nAChR subtypes. loca2 and loca4mRNA are much less abundant and have distinctly differentexpression patterns, suggesting that they do not form subunitsof nAChR subtypes containing a1, a3, and b1 subunits. Tofurther substantiate the subunit composition of locust nAChRsubtypes, immunoisolation of these nAChR using subunit-spe-cific antisera is suggested.

Comparison of the deduced amino acid sequences of thelocust nAChR subunits with those of other insect species indi-cate distinct subfamilies of nAChR isoforms as follows: 1)Loca1 and MARA1 (and the a1 subunit of the lepidopteranHeliothis virescens which was recently cloned in our laborato-ry)2; 2) Loca2 and SBD; 3) Loca3, ALS, and Mpa2; 4) Locb1 andARD; and 5) SAD and aL1. In a phylogenetic tree (Fig. 5) thatwas constructed on the basis of these data, each of the fivesubfamilies conforms to a separate evolutionary branch. Takentogether, these data argue against simplified evolutionary con-siderations such as that because Lipidoptera and Diptera ap-parently are more closely related to each other than to Orthop-tera, so must be their proteins and nucleic acids. Similarly, thepresent data do not suffice to group the nAChR isoforms ac-cording to their physiological function(s) or pharmacologicalprofiles.

Recent studies from several laboratories (47)3 have identifiednicotinic acetylcholine receptors to be involved in cellular ac-tivities other than neurotransmission. Thus, the human a3nAChR expressed in skin keratinocytes has been reported toregulate cell adhesion and motility. The expression of Loca3 incells of the somatogastric epithelium may suggest similar non-neuronal activity.

Expression of nAChR Subunits in the Locust and in EctopicExpression Systems—Transcription of all L. migratoria nAChRsubunits begins at about the same time in late embryonicdevelopment (3 days before hatching) and remains throughoutadulthood. In Drosophila, transcripts of the a and non-a geneswere detected beginning from mid-aged embryos, and the levelsof expression were also developmentally regulated, as observedhere for the locust (for a review, see Ref. 2). As already dis-cussed above, the spatial and temporal expression patternssuggest the formation of several functionally distinct nAChRsubtypes.

The availability of four full-length cDNA clones (three a andone b subunit) with attached signal sequence of a vertebrateneuronal nAChR should offer excellent conditions for the func-tional expression of the Locusta nAChR subtypes. Unfortu-nately, our attempts to express single subunit cDNAs or mix-tures of subunit DNAs in the frog oocytes so far were notsuccessful, even though these studies were performed in col-laboration with research groups that are well experienced inthis area (31, 48, 49). Our unsuccessful attempts reinforceprevious findings that expression of insect nicotinic receptorsin Xenopus oocytes is very difficult to achieve (19). This may bedue to inappropriate assembly of insect receptors in this ectopicexpression system (2, 19), or to missing or inappropriate post-translational modifications, or to the existence of as yet uni-

2 S. Jafari-Gorcini and A. Maelicke, manuscript in preparation.3 A. D. J. Maus, E. F. R. Pereira, K. Macklin, P. I. Karachunski, R. M.

Horton, D. Navaneetham, W. S. Cortes, E. X. Albuquerque, and B. M.Conti-Fine, submitted for publication.

cell bodies; m, mesenchyme. Dashed lines indicate the approximate borders between ganglionic cell bodies and neuropil in the protocerebral loop.Bars, A, 160 mm; B–F, 40 mm; G and H, 20 mm. In ganglionic cells only b1 mRNA (F) but no mRNA coding for a isoforms was expressed, suggestingthat expression of the structural subunit may precede formation of functional channels of a and b subunits.

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dentified (structurally unique) additional subunit(s). We pres-ently attempt to functionally express Locusta cDNAs in amammalian cell line in which vertebrate neuronal nAChRshave been successfully expressed (46). Alternatively, shouldinsect receptors not well express in vertebrate expression sys-tems, the locust receptors may be transiently expressed inDrosophila S2 cells. Functional expression of insect nAChRs incell systems that are suited for electrophysiological studies willbe essential for the elucidation of the molecular basis for thepeculiar pharmacology of some insect receptors (9, 10, 17).Based on the present study, the unusual pharmacology of thelocust nAChR may be brought about by the presence of severalreceptor subtypes in the same cell. Elucidation of the subunitcompositions of functional locust nAChRs and of their subtype-specific pharmacology will therefore remain pressing tasks. Inaddition, such studies might foster the development of novelinsecticides.

Acknowledgments—We thank Eckart Gundelfinger (Magdeburg) andHeinrich Betz (Frankfurt) for generously providing us the ARD2 cDNAclone which we used to prepare the initial screening probe. We thankDaniel Bertrand (Geneva) for help in expressing in Xenopus oocytescDNA mixtures of L. migratoria clones with vertebrate neuronal bclone. We thank Marc Ballivet (Geneva) for providing the rat a3 cDNAclone for the introduction of rat signal sequences into L. migratoriacDNA clones. We thank Sybille Engels for help in the preparation oftissue and embryo sections. We thank Dr. August Dorn (Institute ofZoology, University Mainz) for providing live adult locusts and Dr.Heinz Breer (Institute of Zoology, Stuttgart-Hohenheim; Germany) forproviding muscle and ganglia tissue from adult locusts. The help of PaulSchloder (Institute of Zoology, Mainz) in interpreting the in situ hybrid-ization data is gratefully acknowledged. We thank Anne Rohrbacherand Vesna Pondeljak for perfect technical assistance and MichaelPlenikowski for graphic service. Fruitful discussions with the lateMartin Rentrop and with B. Wieland Kruger (BAYER AG) are grate-fully acknowledged.

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Reinhardt and Alfred MaelickeSchrattenholz, Ulrich Ebbinghaus, Axel Kretschmer, Christoph Methfessel, Sigrid

Bernhard Hermsen, Eva Stetzer, Rüdiger Thees, Reinhard Heiermann, AndreEXPRESSION

: CLONING ANDLocusta migratoriaNeuronal Nicotinic Receptors in the Locust

doi: 10.1074/jbc.273.29.183941998, 273:18394-18404.J. Biol. Chem. 

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