Neuronal Nicotinic Receptors in the Locust Locusta migratoria
CLONING AND EXPRESSION*

Bernhard HermsenDagger , Eva StetzerDagger , Rüdiger TheesDagger , Reinhard HeiermannDagger , Andre SchrattenholzDagger , Ulrich Ebbinghaus§, Axel Kretschmer§, Christoph Methfessel§, Sigrid ReinhardtDagger , and Alfred MaelickeDagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We have identified five cDNA clones that encode nicotinic acetylcholine receptor (nAChR) subunits expressed in the nervous system of the locust Locusta migratoria. Four of the subunits are ligand-binding alpha  subunits, and the other is a structural beta subunit. The existence of at least one more nAChR gene, probably encoding a beta  subunit, is indicated.

Based on Northern analysis and in situ hybridization, the five subunit genes are expressed. localpha 1, localpha 3, and locbeta 1 are the most abundant subunits and are expressed in similar areas of the head ganglia and retina of the adult locust. Because Localpha 3 binds alpha -bungarotoxin with high affinity, it may form a homomeric nAChR subtype such as the mammalian alpha 7 nAChR. Localpha 1 and Locbeta 1 may then form the predominant heteromeric nAChR in the locust brain. localpha 4 is mainly expressed in optic lobe ganglionic cells and localpha 2 in peripherally located somata of mushroom body neurons. localpha 3 mRNA was additionally detected in cells interspersed in the somatogastric epithelium of the locust embryo, suggesting that this isoform may also be involved in functions other than neuronal excitability. Transcription of all nAChR subunit genes begins approximately 3 days before hatching and continues throughout adult life.

Electrophysiological recordings from head ganglionic neurons also indicate the existence of more than one functionally distinct nAChR subtype. Our results suggest the existence of several nAChR subtypes, at least some of them heteromeric, in this insect species.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

In insects, neuromuscular transmission is mediated by glutamate, whereas acetylcholine is the principal neurotransmitter in the nervous system (1). A large body of evidence suggests the existence of both muscarinic and nicotinic acetylcholine (nAChR)1 receptors in the insect brain, with nAChR-coding RNAs having been identified in several species, including the fruit fly Drosophila (2), the locust Schistocerca (3), the tobacco hornworm Manduca (4), and the peach-tomato aphid Myzus (5). Considerable pharmacological diversity of nicotinic receptors is indicated by the existence of alpha BTX-sensitive and -insensitive receptors (6, 7) and by the rather wide variation of responses to nicotinic and non-nicotinic drugs of insect neurons and membrane preparations (8, 9). In particular, the nAChR of Locusta migratoria was suggested to have mixed nicotinic and muscarinic pharmacology (10), which could correlate with the greater evolutionary age of orthoperians as compared with dipterians.

Vertebrate neuronal nicotinic receptors are quite diverse (11), with to date eight alpha  subunits and three beta  subunits cloned in the rat. Of these, the alpha 7, alpha 8, and alpha 9 subunits have the unique ability to form functional homomeric receptors (12-14). Various combinations of the other alpha  and beta  subunits also give rise to functional receptors, as is exemplified by combinations of alpha 4 and beta 2 subunits and of alpha 3 and beta 4 subunits expressed in hippocampal neurons (15). The stoichiometries of heteromeric neuronal nAChRs are not yet established.

The homo-oligomeric receptors appear to be the evolutionarily oldest (16), which has led to the suggestion that the invertebrate neuronal nAChR from L. migratoria, given its broad pharmacology, may be an alpha 7-like homo-oligomeric receptor (17, 18). In Drosophila, five different putative nAChR subunits have been identified, three of which contain the two adjacent cysteines that are characteristic of ligand-binding alpha  subunits (2).

The subunit compositions and stoichiometries of insect nicotinic receptors are still unknown. This is in part due to the fact that expression in Xenopus oocytes of insect nAChR subunit RNA and cDNA has generally proven to be difficult (19). For the same reason, it has not yet been possible to determine the electrophysiological and pharmacological properties of single subtypes of insect nicotinic receptors.

In the present study, we show that in the locust L. migratoria at least six different genes exist that encode nAChR subunits. Four of these genes encode for alpha  subunits. Although we were unable to demonstrate heterologous functional expression in Xenopus oocytes of single subunits, or combinations of subunits, in situ hybridization studies show that the identified subunits are expressed in vivo and probably form functional receptors with different quarternary structures. Because insect nAChRs represent important targets for insecticides (20, 21), the structural and functional characterization of such receptors may be useful in the context of rational drug design.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Locust eggs were supplied by Futtertierversand Hintze, Berlin (Germany). Dr. August Dorn (Institute of Zoology, University Mainz) provided live adult locusts, and Dr. Heinz Breer (Institute of Zoology, Stuttgart-Hohenheim; Germany) provided muscle and ganglia tissue from adult locusts.

Preparation of a Locust-specific Genomic nAChR Probe-- A L. migratoria genomic library was constructed using the lambdoid phage NM1149 in combination with the hfl mutant Escherichia coli strain POP13b, which suppresses the lytic cycle of non-recombinant phages. Chromosomal DNA was prepared from muscle tissue of adult locusts as described (22). DNA was restricted with EcoRI, fractionated on a 0.7% agarose gel, and fragments from 3.8 to 5.4 kilobase pairs length were isolated. Samples of 100 ng of fractionated DNA were ligated with 1 µg of EcoRI-restricted NM1149-DNA and packaged. Packaging efficiency was 8 × 105 lytic plaques per 100 ng of Locusta DNA.

The E. coli strain POP13b was infected with 1.5 × 106 recombinant plaques, and the genomic library was screened using a [alpha -32P]dATP randomly labeled 650-bp fragment from the Drosophila ARD cDNA (23). Filters were hybridized overnight and washed with low stringency (2× SSC, 55 °C; 20× 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. Two recombinants were isolated and characterized. A 177-bp AluI fragment of one of these recombinants coded for the full second transmembrane domain (TM2), the following short loop, and part of the third transmembrane domain (TM3) of a nAChR. This fragment was used as screening probe in further experiments.

Isolation of cDNA Clones-- A L. migratoria cDNA library was constructed in lambda gt11. Poly(A)+ RNA was extracted from ganglionic tissue and reverse-transcribed to double strand DNA using hexanucleotide primers. cDNA was ligated to EcoRI linkers and cloned into lambda gt11. E. coli strain BHB2600 was infected 5.5 × 106 recombinants. 600,000 phages were plated with high density (50,000 plaques/13-cm dish). Screening was performed using randomly primed [alpha -32P]dATP-labeled 177-bp AluI fragment. Filters were washed under low stringency (2× SSC, 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 Localpha 1 cDNA Clone-- RNA was prepared from d9 embryos, and single strand cDNA was prepared from 5 µg of total RNA using a mixture of oligo(dT)12-18 and random primers. Reaction was carried out using 400 units of Superscript Reverse Transcriptase (Life Technologies, Inc.) following the provider's protocol.

1 ng of single strand cDNA was amplified in a first polymerase chain reaction (PCR) using 3'-primers highly specific for Localpha 1 (5'-CTCGAGGGACATGTAGAACTCGGAGAGGTC-3') and a mix of two 5'-primers (5'-ACCGCCTCATCAGGCCTGTCACCAACAACTCCGA-3' and 5'-ACCGCCTCATCAGGCCTGTCGGCAACAACTCGGA-3') designed to highly homologous regions of localpha 2 and localpha 3 cDNA clones (aa 15-21 of localpha 2 and aa 4-10 of localpha 3, respectively). Both 5'-primers contained a silent mutation to introduce a StuI site (bold letters) which was later used to fuse the rat alpha 3 nAChR signal peptide to the locust alpha 1 clone. PCR products were diluted 1:100 and re-amplified in a second PCR using the same 5'-primers and another 3'-primer (5'-GGCAGGCACCTCGAGGATGTCCCA-3') designed to the already amplified fragments. Both PCRs were carried out in a volume of 20 µl under the following conditions: 1 × Taq buffer (Life Technologies, Inc.), 10 pM of each primer, 5 mM MgCl2, 200 µM dNTP, 50 mM Tris-HCl. pH 9.5, 2.5 units of Taq Polymerase (Life Technologies, Inc.). After incubation at 94 °C, 5 min amplification was carried out in a Perkin-Elmer Thermocycler 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 into pSL1180 vector (Amersham Pharmacia Biotech). Clones containing localpha 1-specific sequences were isolated by colony hybridization (Sambrook et al., 1989) using a [alpha -32P]dATP-labeled oligo (5'-CTCGAGGGACATGTAGAACTCGGAGAGGTC-3'), which is highly specific for localpha 1. They were then sequenced, and one of them was fused to the partial localpha 1 cDNA clone.

Construction of Full-length cDNAs-- The BamHI/EcoRI fragment of the rat alpha 3 nAChR cDNA (24) and the locust alpha 2, alpha 3, and beta  cDNAs were cloned into pBluescript® KS+ (Stratagene). A StuI site was introduced into the 5'-end of these clones by PCR without changing the Arg/Pro amino acid sequence at this site. PCR was performed with 1 ng of each clone under the same conditions as described above. Amplification of the locust clones was carried out using a 3'-primer, designed to the pBluescript® KS+ sequence (5'-AACAGCTATGACCATG-3'), and the following specific 5'-primers containing a silent mutation (in order to introduce a StuI site; see in bold letters) (localpha 2, 5'-ACCGCCTCATCAGGCCTGTCACCAACAACTCCGA-3'; localpha 3, 5'-ACCGCCTCATCAGGCCTGTCGGCAACAACTCGGA-3'; locbeta , 5'-ACAAGCTCATCAGGCCTGTGCAGAACATGACGCA-3'). Amplification of the rat alpha 3 nAChR fragment was carried out with 2 ng of cloned DNA using the following primers: 5'-GTAAAACGACGGCCAGT-3', 5'-CCTCCGAATTCTCCGGA-GATGATCTCGTTGTAAT-3'). PCR products were cloned into pBluescript® KS+ and sequenced. The StuI site was then used to fuse the rat alpha 3 nAChR signal sequence to each of the locust cDNA clones.

In Situ Hybridization-- In situ hybridization was performed with digoxigenin-labeled RNA probes. RNA probes were randomly labeled with digoxigenin according to the protocol provided by the manufacturer (Boehringer Mannheim, Germany). In order to obtain isoform-specific probes, sequence regions with lowest homology were selected, i.e. for localpha 1 and localpha 2 clones from the "cytoplasmic loop" between TM3 and TM4, for locbeta from parts of the 5'-translated region, for localpha 3 from the 3'-untranslated region, and for localpha 4 from the 5'-untranslated region. The isoform specificity of the RNA probes was confirmed by Southern analysis.

Whole embryos and head ganglia with attached optic lobes and retina from adult locusts were dissected out of the eggshell and individual adult insect, respectively, placed in embedding medium (Tissue Tec, Miles), and shock-frozen with dry ice. They were kept at -70 °C until use. 6-9-µm frozen sections were obtained at -20 °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, 5× SSC, 50 µg/ml tRNA, 50 µg/ml heparin, 0.1% Tween 20) for 1 h at 50 °C. Digoxigenin-labeled RNA probes were added, and the cryosections were incubated overnight. After five washes for 20 min in 2× SSC, 50% formamide, and treatment with RNase A (25 mg/ml) and RNase T1 in 2× SSC, 8 further washes were performed in which the SSC buffer was diluted out with PBS-T in a stepwise fashion. After incubation for 1 h with 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 (Boehringer 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 dye reagents nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After washing the sections for 5 min in PBS, they were covered in PBS, 50% glycerol and were analyzed under the microscope.

Southern and Northern Blot Analysis-- 10 µg of chromosomal DNA prepared from muscle tissue of adult locusts was completely restricted with EcoRI, BamHI, and HindIII, fractionated, and blotted onto a nylon membrane. The genomic blot was hybridized with the randomly primed [alpha -32P]dATP-labeled AluI fragment and was washed under moderate stringency (65 °C; 1× SSC).

RNA was prepared from embryos d4-d9 after egg laying. 1 mg of RNA was used to prepare poly(A)+ RNA. Every 5 µg of poly(A)+ RNA of all six developmental stages was submitted to electrophoresis and blotted onto a nylon membrane. Blots were hybridized with three random primed [alpha -32P]dATP-labeled probes: a 420-bp HinfI/HinfI fragment of the localpha 2 cDNA, a 230-bp EcoRI/SphI fragment of the locbeta 1 cDNA, and a 370-bp EcoRI/SphI fragment of the localpha 1 cDNA. Blots were washed with 1× SSC, 65 °C.

Fragments of nAChR alpha  Subunits Obtained by Expression in E. coli and Binding Studies with alpha -Bungarotoxin-- cDNA clones were obtained from the following sources: Drosophila alpha 1 from Marc Ballivet, Geneva, Switzerland (25); Drosophila alpha 2 from Eckart Gundelfinger, Magdeburg, Germany (26); and Torpedo alpha  from Toni Claudio, New Haven, CT. cDNA fragments of the N-terminal extracellular regions were expressed either as maltose-binding protein fusions using pMAL-c2 vector from New England Biolabs (Schwalbach, Germany) or as glutathione S-transferase fusions using pGEX-4T-1 vector (Amersham Pharmacia Biotech). Fusion proteins were prepared from isopropyl-1-thio-beta -D-galactopyranoside-induced E. coli transformants (E. coli strain DH5alpha , Life Technologies, Inc.) of flask submersed cultures. E. coli pellets were sonicated in lysis buffer, and after centrifugation, the supernatant was diluted and applied to affinity chromatography. Eluted nAChR fusion proteins were dialyzed, and the purity was determined by capillary electrophoresis and SDS-PAGE. Based on these methods, the purity of fusions proteins was generally better than 85%. In selected examples (Torpedo alpha  fragment), the fusion protein was proteolytically cleaved (factor Xa), and the nAChR fragment was isolated, and terminal peptide sequencing and mass spectrometry were performed. These data confirmed the primary structure of the nAChR fragments. Fusion proteins were separated on a, SDS-polyacrylamide gel, which was then blotted onto a nitrocellulose membrane. The membrane was incubated with 10-9 M 125I-alpha BTX at 4 °C, 30 min, and washing was performed 5 × for 30 min with 1× PBS. 125I-alpha BTX was obtained from Amersham Pharmacia Biotech (Braunschweig, Germany).

Preparation of Locust Neurons and Electrophysiological Recordings-- Head (supra-esophageal) ganglia and optic lobes from individual adult L. migratoria were dissected out and placed into dissociation solution (Sigma-Aldrich, Deisenhofen, Germany). Dispase (2 mg/ml, Life Technologies, Inc., Eggenstein, Germany) was added and incubated for 5 min at 37 °C. The material was then centrifuged, and the pellet was resuspended in culture buffer and was dissociated by gentle aspiration with a fire-polished Pasteur pipette (27). Cells were plated onto glass coverslips that were pre-coated with concanavalin A (400 µg/ml, Sigma) and laminin (4 µg/ml, Sigma). The cultures were kept at room temperature and used for electrophysiological measurements on the following 2 days.

For electrophysiological recordings, the whole cell patch clamp technique was used. Microelectrodes were pulled from borosilicate glass capillaries (Hilgenberg, Malsfeld, Germany). The resistance of the fire-polished pipettes was 4-7 megohms, using the internal and external solutions described below. All experiments were performed at room temperature (22-25 °C).

Cells were placed in a perfusion chamber at approximately 0.5-ml volume and superfused continuously (flow rate 3 ml/min) with external bath solution. The bath solution contained (in mM): 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, pH 7.4, adjusted with NaOH. The (intracellular) pipette solution contained (in mM): 120 CsF, 30 CsCl, 10 Cs-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 -70 mV. Current records were low pass Bessel filtered at 315 Hz and digitized at 1-kHz sample rate. Data storage and analysis were performed with the pClamp version 6.03 software package (Axon Instruments, Foster City, CA). Test substances were applied to the cells using the U-tube reversed flow technique (28) with applications of 1-2 s duration at intervals of 1 min. Acetylcholine chloride, cytisine, and coniine were obtained from Sigma. Nicotine bitartrate was obtained from RBI (Natick, MA). Drugs were stored frozen as stock solutions (10 or 100 mM in water) and thawed and diluted on the day of the experiment.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Electrophysiological Studies Suggest Several Subtypes of Neuronal nAChR in the Locust L. migratoria-- The dissociated neurons obtained from head (supra-esophageal) ganglia and optic lobes of L. migratoria and cultured for 1-2 days on glass coverslips were large, round cells between 30 and 120 µm in diameter, frequently having short protrusions. These cells invariably responded to test applications of 10 µM acetylcholine (ACh) with a fast inward current of between 100 and 2000 pA at -70 mV clamp potential. Dose-response curves obtained with ACh on head ganglion cells (Fig. 1) yielded an EC50 value of 17.9 ± 3.4 µM (n = 7), whereas an EC50 of 19.1 ± 8.9 (n = 4) was measured for optic lobe cells (not shown). In both cases, other nicotinic agonists such as (-)-nicotine or cytisine elicited responses that, even at saturating concentrations, remained well below the maximal currents induced by ACh.


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Fig. 1.   Current responses of a neuron from the locust head ganglion to applications of acetylcholine at -70 mV membrane potential. The dose-response curve was fitted by the Hill equation. All currents were normalized to the mean amplitude elicited by 10 µM ACh before and after each test concentration was applied. The EC50 for ACh was 17.9 ± 3.4 µM (mean ± S.D., n = 7 cells). The upper inset shows typical ACh responses at different concentrations.

The time course of the response to ACh was quite variable from one cell to another. In addition to the rapidly desensitizing component present in most cells, some cells also exhibited a non-desensitizing response to ACh, suggesting the existence of (at least) two nAChR subtypes. Further evidence was provided by dose-response curves for nicotine on cells in which one or the other type of ACh response predominated (Fig. 2). In a cell with mostly rapidly desensitizing ACh-induced currents, nicotine was a weak partial agonist with EC50 >20 µM (29). By contrast, another cell with non-desensitizing ACh currents responded sensitively to nicotine, with an EC50 of <1 µM. Since most cells contained both subtypes of nAChR in varying proportions, the effective EC50 values for nicotine were quite variable (e.g. 9.4 ± 11.4 µM, n = 5 in optic lobe cells). Similar results were obtained with cytisine, whereas coniine acted as agonist exclusively at the non-desensitizing nAChR subtype. Taken together, these data confirm the existence of functional nAChR ion channels in locust neurons and clearly suggest that there exist at least two pharmacologically distinct subtypes of nAChR in the cells studied.


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Fig. 2.   Current responses of two ganglion cells to ACh and nicotine, and dose-response curves for nicotine. Upper, current responses of two different locust ganglion cells to ACh and nicotine at -70 mV membrane potential. Note the very different response amplitudes to nicotine. Lower, dose-response curves for nicotine taken from the same two cells. The current values were normalized to the maximal current elicited by a saturating concentration of ACh on each cell.

The L. migratoria Genome Contains Several Genes Encoding for nAChR Subunits-- A genomic library was prepared from muscle tissue of adult L. migratoria nAChR and was screened with a 32P randomly labeled 650-bp long fragment from Drosophila ARD2 cDNA (23) encoding the transmembrane regions TM1-TM3. We obtained among other clones a 4.1-kilobase pair long genomic fragment that displayed 69.2% homology to the Drosophila ARD2 sequence, 81.6% to the Drosophila ALS sequence, and 75.9% to the chick alpha 4 sequence. An AluI fragment (177 bp) of the genomic clone, containing mostly coding sequences (140 bp) and encoding the full second transmembrane domain (TM2), the following short loop, and part of the third transmembrane domain (TM3), was prepared, randomly labeled with [32P]dATP, and used as probe in a genomic blot of purified DNA from muscle tissue of adult locusts that was cleaved with the restriction endonucleases EcoRI, HindIII, and BamHI. As shown in Fig. 3, at least six bands were detected in each lane, suggesting that this number represents the approximate number of nAChR-coding genes that exist in L. migratoria.


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Fig. 3.   Southern blot analysis of L. migratoria chromosomal DNA treated with three different restriction endonucleases. L. migratoria chromosomal DNA was obtained from muscle tissue. 10 µg of DNA were treated with the restriction enzymes EcoRI (E), HindIII (H), and BamHI (B), respectively, fractionated, and transferred onto a nylon membrane. The blot was hybridized under low stringency conditions with a 32P-labeled genomic probe of 177 bp, containing mostly coding sequences (140 bp) and encoding the full second transmembrane domain (TM2), the following short loop, and part of the third transmembrane 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.

Isolation and Sequencing of Five cDNAs Coding for nAChR Subunits-- After initial information was obtained by Northern analysis (see later for details) for the developmental stages at which nAChR subunit mRNA is expressed in the locust, we prepared a cDNA library from late embryonic stage. By using the 32P-labeled genomic AluI fragment described above, we screened the cDNA library at relatively high stringency. We obtained several cDNA clones which coded for four alpha  subunits and one beta  subunit. The cDNA clones localpha 2, localpha 3, and locbeta 1 encoded for mature subunits, whereas clone localpha 1 missed the nucleotides encoding for the first approximately 200 amino acids of the mature protein. All four cDNA clones missed the 5'-terminal sequences encoding the signal sequences. Another cDNA clone isolated encoded the full N-terminal sequence of an alpha  subunit (localpha 4) but extended only to the end of transmembrane domain 2. The complete sequence of localpha 1 was obtained by PCR. Attempts to also obtain by PCR the signal sequences of the four full-length cDNA clones were not successful. The mature proteins of L. migratoria Localpha 1-alpha 3 subunits consist of 559, 515, and 540 amino acids, respectively, with predicted molecular masses of 61.5, 56.7, and 59.4 kDa. The Locbeta subunit consists of 497 amino acids with a molecular mass of 54.7 kDa. The nucleotide sequences of the four full-length clones have been reported to the EMBO nucleotide sequence Data Bank (accession numbers AJ000390-000393). Since none of the five cDNAs was identical in sequence to the exon sequence of the genomic AluI fragment used as screening probe (homologies are 79-89% for the alpha  clones, 64% for the beta  clone), there must exist at least one more gene in L. migratoria that encodes nAChR subunits.

Properties of Sequences and Homology to Other Insect nAChRs-- In Fig. 4 (upper panel), the hydropathy plot of the Localpha 2 isoform is representatively shown. As is typical of all isoforms of nAChR identified, it displays the pattern of four putative transmembrane domains (TM1-4) that is common for the superfamily of ligand-gated ion channels. The sequences coding for the alpha -isoforms also contain in their N-terminal extracellular domain the four conserved cysteines (Fig. 4, lower panel), including the two vicinal ones just in front of the first transmembrane domain, and two putative N-glycosylation sites. The cytoplasmic domain between TM3 and TM4 is quite variable in size (199, 147, 185, and 156 amino acids in Localpha 1-3 and Locbeta 1, respectively) and contains several putative phosphorylation sites. In the Locbeta 1 sequence, this region contains a putative phosphorylation site for cAMP-dependent kinase, as was also found in non-alpha subunits from Torpedo (30) and Drosophila (23).


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Fig. 4.   Hydropathy profile of Localpha 2 subunit and generalized domain structures of L. migratoria nAChR subunits. The amino acid residues are numbered according to the Localpha 2 sequence (Fig. 4). M1-M4 refer to putative transmembrane domains.

Sequence homologies of the four full-length clones of the L. migratoria nAChR subunits to those of other species are reported in Fig. 5, top and bottom.


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Fig. 5.   Phylogenetic tree of insect nAChR isoforms. The phylogenetic tree (top) was constructed on the data presented in the table (bottom). Amino acid identity between complete amino acid sequences except the cytoplasmic loop was obtained using Clustal method with PAM250 weight table. Accession numbers are as follows: ALS, P09478; Dalpha 2/SAD, P17644; ARD, X04016; SBD, X55676; Mpalpha 1, X81887; Mpalpha 2, X81888; alpha L1, P23414; MARA1, Y09795. NAChR isoforms with 80%, or more, amino acid sequence identity to particular Loc subunits are indicated in bold in the table. These subfamilies each represent a separate evolutionary branch.

Attempts to Express the L. migratoria nAChR cDNAs and mRNA in Xenopus Oocytes-- Functional expression in Xenopus oocytes of insect nAChR following injection of mRNA into the cells has been reported for the cloned alpha  subunit from the locust Schistocerca gregaria (3). Functional channels were formed that were gated by micromolar concentrations of nicotine and that were blocked by alpha BTX, kappa BTX, strychnine, and bicuculline. These results suggest that in Schistocerca there exists a homomeric nAChR that has similar physiological properties as the nicotinic responses recorded from insect neurons. In contrast, Drosophila nAChR alpha  subunits so far could only be co-expressed in Xenopus oocytes with the chick neuronal beta 2 subunit (31).

We have undertaken various attempts, so far in vain, to express the cloned L. migratoria nAChR subunits in Xenopus oocytes. To achieve expression, we introduced by silent mutagenesis a StuI restriction site at the 5'-end of each clone, and by using this site, we attached rat alpha 3 signal sequence to the Locusta cDNA clones. We then injected into Xenopus oocytes single subunit cDNA and cDNA mixtures of different subunits or in vitro transcribed mRNA, neither treatment led to any significant channel activity. We also were unable to reproduce for any of the localpha clones the successful co-expression in Xenopus oocytes of Drosophila alpha  subunit with the vertebrate beta 2 clone (31). The inability of the cloned Locusta subunits to form functional channels in Xenopus oocytes may be due to inappropriate assembly of insect receptors in this ectopic expression system (2, 19), or to missing or inappropriate post-translational modifications (32, 33), or to additional as yet unidentified subunits that are required for assembly and/or channel function. That functional ion channels are formed from L. migratoria nAChR subunits was shown by Hanke and colleagues (18, 34) who reported electrophysiological recordings from affinity purified and reconstituted nAChR protein.

The Localpha 3 nAChR Isoform Binds alpha -Bungarotoxin-- Binding of the snake neurotoxin alpha BTX to L. migratoria nAChR isoforms was studied by Western blotting. For this purpose, fusion proteins between either maltose-binding protein or glutathione S-transferase and fragments of nAChR alpha  subunits were expressed in E. coli, and the purified fusion proteins were applied to Western blotting using as probe 125I-labeled alpha BTX. As is representatively shown in Fig. 6, the Localpha 3 fusions (containing aa 126-229 and aa 12-227, respectively) and (as controls) the Torpedo alpha  fusion (aa 1-246) and Drosophila alpha 1 (ALS) fusion (aa 83-223) bound alpha BTX, whereas the Localpha 2 fusion (aa 1-222) and the Drosophila alpha 2 fusion (aa 3-226) did not bind I-alpha BTX. To exclude the possibility of nonspecific binding of alpha BTX to the fusion partner, nAChR fragments were released by proteolytic cleavage from fusion proteins and were then tested in Western blots for toxin binding. These experiments (not shown) confirmed the above findings. In other Western blotting experiments (not shown), binding of alpha BTX to Localpha 3 (aa 12-290), Torpedo alpha 1 (aa 143-201), rat alpha 1 (aa 1-210), and rat alpha 7 (aa 80-213) was demonstrated. Selective binding of alpha BTX by Localpha 3, as compared with Localpha 2, cannot be explained by differences in sequence in the region around the two adjacent cysteines of alpha  subunits but rather appears to be due to attachment points for the toxin that are located in other sequence region(s). That the binding site for alpha BTX is discontinuously distributed within the N-terminal region of the alpha  subunit has previously been reported for nAChRs from other species (34).


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Fig. 6.   Binding of alpha -bungarotoxin to fusion proteins containing fragments of the N-terminal extracellular region of nAChR alpha  subunits from several species. Samples of affinity purified fusion proteins containing nAChR fragments from Torpedo alpha  (aa 1-246, lane 1), Drosophila alpha 2 (aa 3-226, lane 2), Loc alpha 3 (aa 126-229, lane 3), Loc alpha 3 (aa 12-227, lane 4), Loc alpha 2 (aa 1-222, lane 5), and Drosophila alpha 1 (aa 83-223, lane 6) were separated on SDS-PAGE and stained with Coomassie (upper) or blotted onto nitrocellulose for Western blotting using 10-9 M mono-iodinated 125I-alpha BTX (bottom). The complex pattern of protein bands in lanes 4 and 5 is probably caused by premature translation stop of malE-nAChR fusion polypeptides. Binding of alpha BTX was detected for Torpedo-alpha , Localpha 3, and Dalpha 1.

From its alpha BTX binding properties, Localpha 3 is a candidate for a homo-oligomeric nAChR, such as the alpha 7 subtype of mammals (11, 35). As reported above though, we have been unable so far to functionally express this or other isoforms in Xenopus oocytes, as was achieved for other homo-oligomeric nAChR (13, 36). We therefore do not know whether the Localpha 3 isoform indeed forms a functional channel with the typical properties of homo-oligomeric nAChRs, e.g. fast desensitization and sensitivity to epibatidine and choline (37).

Western blotting with the same fusions proteins was also performed with the antibody WF6 which competes with ACh and competitive agonists and antagonists (including alpha BTX) for binding to the Torpedo nAChR (34, 38, 39). Selective binding of WF6 was observed only to the Localpha 3 fusion proteins and (as controls) to those from Torpedo alpha  and Dalpha 1 (data not shown). These observations agree with previous findings in that the attachment point patterns within the binding sites for alpha BTX and WF6 seem to be overlapping, albeit distinct (40).

Temporal and Spatial Expression of L. migratoria nAChR mRNA in the Developing and Adult Insect-- Initial information on developmental stage-specific expression of L. migratoria mRNA was obtained by Northern analysis (Fig. 7). We employed randomly labeled 32P-cDNA probes that were selected on the basis of minimal sequence homology between each other and that did not cross-react with the other cDNA clones, as was tested by Southern analysis (not shown). As is representatively shown for localpha 1, localpha 2, and locbeta 1 in Fig. 7, the nAChR subunit RNAs begin to be expressed in late embryonic development, i.e. around d7 after egg laying and 3 days before hatching of these hemi-metabolic insects. Expression increased and reached a maximal level approximately 1 day before hatching. Northern blots probed with the beta  cDNA probe always showed two transcripts (6.1 and 4.1 kilobase pairs), suggesting cross-hybridization with a second (as yet unidentified) beta  subunit mRNA.


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Fig. 7.   Developmental stage-specific expression of L. migratoria nAChR subunits alpha 1, alpha 2, and beta 1 as studied by Northern blot analysis. 5 µg of poly(A)+ RNA of each embryonic day (d4-9) were submitted to electrophoresis and blotted onto a nylon membrane. Hybridization was performed with randomly 32P-labeled cDNA fragments of clones localpha 1, localpha 2, and locbeta 1. A, 420-bp long HinfI/HinfI fragment from localpha 2; B, 370-bp long EcoRI/SphI fragment from localpha 1; C, 230-bp long EcoRI/SphI fragment from locbeta 1. Size markers were mouse rRNA (28 S and 18 S). All three blots show beginning subunit mRNA expression at day 6 after egg laying, with maximal expression on d8.

Temporal and spatial expression of L. migratoria nAChR mRNA was studied in further detail by in situ hybridization with subunit-specific RNA probes of frozen sections of 6-9 µm thickness. In the scheme of Fig. 8A, the areas are indicated in which nAChR mRNA was detected in the adult locust; these are the two-paired head ganglia, called mushroom bodies, the paired optic lobes, and the retina. The mushroom bodies are composed of large neurons, the cell bodies of which are located peripherally, whereas the neurites form the central neuropil. The cell bodies of the optic lobe neurons are clustered in a structure that is close to the retina (see also Fig. 9A).


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Fig. 8.   Expression of nAChR isoforms in head ganglia of the adult locust. A, schematic representation (modified after Ref. 51) of the anatomic organization of the adult locust head ganglia with the supra-esophageal ganglion (mushroom body, framed) which consists of ganglionic cell bodies (gcb) and neuropil (np), the optic lobes, including the lamina ganglionaris (lg), medulla externa (me), medulla interna (mi), and the optic lobe connective (ole), and the retina (r). The area from which the micrographs B-F were taken is framed. B-F, in situ hybridizations of mRNA encoding for alpha 1 (B), alpha 2 (C), alpha 3 (D), alpha 4 (E), and beta 1 (F) nAChR isoforms using cryostat sections of isolated locust brains. Digoxigenin-labeled antisense-RNA probes were used (for details, see "Experimental Procedures"). p, perineurium. Bars, 40 µm. In the areas depicted, mRNA coding for the subunits alpha 1, alpha 2, alpha 3, and beta 1 was detected in the ganglionic cell bodies (see also inset of C) but not in the neuropil (the approximate borders between 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 retina and the accessory optic lobe, and the cellular component of an ommatidium (modified after Ref. 51). The areas depicted in the in situ hybridizations of C-G are framed. C-G, in situ hybridizations with digoxigenin-labeled RNA probes for alpha 1 (C), alpha 3 (D), alpha 4 (F and G), and beta 1 (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). The abbreviations used are: c, cornea; cc, crystal cone; gcb, ganglionic cell bodies; lg, lamina ganglionaris; me, medulla externa; mi, medulla interna; o, ommatidium; pc, photoreceptor cell; ppc, primary pigment cell; spc, secondary pigment cell. Bars, 20 µm. In the areas depicted, mRNA coding for the subunits alpha 1, alpha 3, and beta 1 was detected in primary and secondary pigment cells of single ommatidia in the retina. In contrast, alpha 4 mRNA was expressed in ganglionic cells of the optic lobe. Dashed lines in G indicate the approximate borders between ganglionic cell bodies and the lamina ganglionaris. Inset, alpha 4 mRNA was located in the perinuclear region of ganglionic cell bodies.

As shown in Fig. 8, B-F, mRNA encoding localpha 1-3 and locbeta 1, but not localpha 4, was detected in the mushroom bodies of the adult locust. The strongest expression was observed for localpha 1 mRNA, in the cytosol of neurons whose cell bodies are located in the center of the mushroom bodies. In roughly the same area, localpha 3 and locbeta 1 mRNA was detected. localpha 3 mRNA was expressed to a much lower extent than localpha 1 or locbeta 1. localpha 4 mRNA was not detected in mushroom bodies. localpha 2 mRNA was more abundant in peripherally located cell somata. Specificity of the hybridizations is indicated by the cellular staining pattern which was confined to the cytosol and did not include the cell nuclei (see inset of Fig. 8C). As a general observation, the neurons containing Locusta nAChR mRNA were intermingled with unlabeled nerve cells, suggesting that not all neurons of the locust express nicotinic receptors. The similar expression patterns of localpha 1, localpha 3, and locbeta 1 suggest that these nAChR subunits may form a single (consisting of all three subunits) or two separate (a homo-oligomeric and a hetero-oligomeric one) receptor subtypes.

Expression of nAChR isoforms in the retina and optic lobes is shown in Fig. 9. In the retina, localpha 1 mRNA was most abundant, followed by locbeta 1 and localpha 3 mRNA. localpha 2 and localpha 4 mRNA were absent. Transcripts of the three subunits were located in primary and secondary pigment cells which surround the crystal cones. Whereas localpha 4 mRNA was absent in the retina (Fig. 9F), it was detected in the ganglionic cell bodies of the lamina ganglionaris of the optic lobes where it was located in cell bodies that are situated close to the fibrous part of the optic lobe (Fig. 9G). As concluded above from the expression patterns in the mushroom bodies, Localpha 1, Localpha 3 and Locbeta 1 may form a single or two separate nAChR subtypes.

As demonstrated in Fig. 10, the expression patterns of nAChR isoforms in the d8 locust embryo are quite different from those in the adult locust. Whereas locbeta 1 mRNA was abundantly expressed in the protocerebral lobe, the developing mushroom body (Fig. 10F), the alpha  subunits were not expressed in this area, to any comparable extent. This suggests that the developing mushroom bodies of embryonic day 8 do not yet express functional nAChR and that pre-expression of a structural subunit may be required for functional assembly of heteromeric locust nAChRs. locbeta 1 mRNA was also detected in the ectoderm anlage from which the eye develops (Fig. 10F).


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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 a beta 1-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 alpha 1 (B), alpha 2 (C), alpha 3 (D and G), alpha 4 (E), and beta 1 (F and H) nAChR isoforms, of cryostat 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 cell bodies; m, mesenchyme. Dashed lines indicate the approximate borders between ganglionic cell bodies and neuropil in the protocerebral loop. Bars, A, 160 µm; B-F, 40 µm; G and H, 20 µm. In ganglionic cells only beta 1 mRNA (F) but no mRNA coding for alpha  isoforms was expressed, suggesting that expression of the structural subunit may precede formation of functional channels of alpha  and beta  subunits.

Outside of the head area, localpha 3 mRNA was mainly detected in isolated cells interspersed in the somatogastric epithelium (Fig. 10G). Additional limited expression of localpha 3 mRNA was observed in peripheral ganglion cells distributed in the mesenchyme below the somatogastric epithelium. In contrast, locbeta 1 mRNA was exclusively found in peripheral ganglionic cells (Fig. 10H).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

At Least Six nAChR Subunits Are Expressed in the Locust-- They form several functionally distinct subtypes. In the course of this study, we have identified in the locust L. migratoria six nAChR subunit genes. Three cDNAs encoding alpha  subunits and one cDNA encoding a beta  subunit were obtained as full-length clones. In addition we have isolated a partial nAChR alpha  subunit cDNA and have identified in Northern blots (Fig. 3) a sixth mRNA which probably encodes another beta  subunit. This is the largest number of nAChR subunit genes so far identified in an invertebrate species, and our results clearly contradict previous suggestions of a single homomeric nAChR in L. migratoria (17). In SDS-PAGE of Torpedo nAChR, the four subunits are better separated than expected from their differences in molecular mass (between 50.2 and 57.6 kDa) (50). In contrast, the gel pattern of the affinity purified Locusta nAChR protein suggested a single polypeptide of approximately 65 kDa (17, whereas the molecular weights calculated from the amino acid sequences vary by approximately the same amount (between 54.7 and 61.5 kDa) as the Torpedo subunits. These differences in SDS-PAGE resolution are probably due to variations in the levels of posttranslational modifications.

Our cloning data clearly suggest that several subtypes of nicotinic receptors exist in the locust. These findings are supported by electrophysiological recordings from head ganglia neurons (Figs. 1 and 2) which indicate at least two pharmacologically distinct nAChR subtypes (9).

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

The spatial expression patterns of the Locusta nAChR mRNAs are consistent with the existence of several receptor subtypes. Localpha 1, Localpha 3, and Locbeta 1 are the isoforms most abundantly expressed in the head ganglia and the retina, whereas Localpha 4 is mainly expressed in optic lobe ganglionic cells. Based on its alpha BTX binding properties (Fig. 6), Localpha 3 is a candidate for a homomeric nAChR. Extending this line of arguments, Localpha 1 and Locbeta 1 may form the predominant hetero-oligomeric nAChR of the locust. In the absence of functional expression studies in Xenopus oocytes or other ectopic expression systems, it cannot be excluded though that Localpha 1, Localpha 3, and Locbeta 1 together form a heteromeric nAChR (42-45). The early presence of Locbeta 1 in the embryo of the locust suggests that its expression may be a prerequisite for functional assembly of some hetero-oligomeric nAChR subtypes. localpha 2 and localpha 4 mRNA are much less abundant and have distinctly different expression patterns, suggesting that they do not form subunits of nAChR subtypes containing alpha 1, alpha 3, and beta 1 subunits. To further substantiate the subunit composition of locust nAChR subtypes, immunoisolation of these nAChR using subunit-specific antisera is suggested.

Comparison of the deduced amino acid sequences of the locust nAChR subunits with those of other insect species indicate distinct subfamilies of nAChR isoforms as follows: 1) Localpha 1 and MARA1 (and the alpha 1 subunit of the lepidopteran Heliothis virescens which was recently cloned in our laboratory)2; 2) Localpha 2 and SBD; 3) Localpha 3, ALS, and Mpalpha 2; 4) Locbeta 1 and ARD; and 5) SAD and alpha L1. In a phylogenetic tree (Fig. 5) that was constructed on the basis of these data, each of the five subfamilies conforms to a separate evolutionary branch. Taken together, these data argue against simplified evolutionary considerations such as that because Lipidoptera and Diptera apparently are more closely related to each other than to Orthoptera, so must be their proteins and nucleic acids. Similarly, the present data do not suffice to group the nAChR isoforms according to their physiological function(s) or pharmacological profiles.

Recent studies from several laboratories (47)3 have identified nicotinic acetylcholine receptors to be involved in cellular activities other than neurotransmission. Thus, the human alpha 3 nAChR expressed in skin keratinocytes has been reported to regulate cell adhesion and motility. The expression of Localpha 3 in cells of the somatogastric epithelium may suggest similar non-neuronal activity.

Expression of nAChR Subunits in the Locust and in Ectopic Expression Systems-- Transcription of all L. migratoria nAChR subunits begins at about the same time in late embryonic development (3 days before hatching) and remains throughout adulthood. In Drosophila, transcripts of the alpha  and non-alpha genes were detected beginning from mid-aged embryos, and the levels of expression were also developmentally regulated, as observed here for the locust (for a review, see Ref. 2). As already discussed above, the spatial and temporal expression patterns suggest the formation of several functionally distinct nAChR subtypes.

The availability of four full-length cDNA clones (three alpha  and one beta  subunit) with attached signal sequence of a vertebrate neuronal nAChR should offer excellent conditions for the functional expression of the Locusta nAChR subtypes. Unfortunately, our attempts to express single subunit cDNAs or mixtures of subunit DNAs in the frog oocytes so far were not successful, even though these studies were performed in collaboration with research groups that are well experienced in this area (31, 48, 49). Our unsuccessful attempts reinforce previous findings that expression of insect nicotinic receptors in Xenopus oocytes is very difficult to achieve (19). This may be due to inappropriate assembly of insect receptors in this ectopic expression system (2, 19), or to missing or inappropriate post-translational modifications, or to the existence of as yet unidentified (structurally unique) additional subunit(s). We presently attempt to functionally express Locusta cDNAs in a mammalian cell line in which vertebrate neuronal nAChRs have been successfully expressed (46). Alternatively, should insect receptors not well express in vertebrate expression systems, the locust receptors may be transiently expressed in Drosophila S2 cells. Functional expression of insect nAChRs in cell systems that are suited for electrophysiological studies will be essential for the elucidation of the molecular basis for the peculiar pharmacology of some insect receptors (9, 10, 17). Based on the present study, the unusual pharmacology of the locust nAChR may be brought about by the presence of several receptor subtypes in the same cell. Elucidation of the subunit compositions of functional locust nAChRs and of their subtype-specific pharmacology will therefore remain pressing tasks. In addition, such studies might foster the development of novel insecticides.

    ACKNOWLEDGEMENTS

We thank Eckart Gundelfinger (Magdeburg) and Heinrich Betz (Frankfurt) for generously providing us the ARD2 cDNA clone which we used to prepare the initial screening probe. We thank Daniel Bertrand (Geneva) for help in expressing in Xenopus oocytes cDNA mixtures of L. migratoria clones with vertebrate neuronal beta  clone. We thank Marc Ballivet (Geneva) for providing the rat alpha 3 cDNA clone for the introduction of rat signal sequences into L. migratoria cDNA clones. We thank Sybille Engels for help in the preparation of tissue and embryo sections. We thank Dr. August Dorn (Institute of Zoology, University Mainz) for providing live adult locusts and Dr. Heinz Breer (Institute of Zoology, Stuttgart-Hohenheim; Germany) for providing muscle and ganglia tissue from adult locusts. The help of Paul Schlöder (Institute of Zoology, Mainz) in interpreting the in situ hybridization data is gratefully acknowledged. We thank Anne Rohrbacher and Vesna Pondeljak for perfect technical assistance and Michael Plenikowski for graphic service. Fruitful discussions with the late Martin Rentrop and with B. Wieland Krüger (BAYER AG) are gratefully acknowledged.

    FOOTNOTES

* This work was supported by the Stiftung für Innovation Rheinland/Pfalz, the Fonds der Chemischen Industrie, and BAYER AG, Leverkusen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" 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 GenBankTM/EMBL Data Bank with accession number(s) AJ000390-000393.

To whom correspondence should be addressed. Tel.: 49 6131 395911; Fax: 49 6131 393536; E-mail: alfred.maelicke{at}uni-mainz.de.

1 The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; alpha BTX, alpha -bungarotoxin; kappa BTX, kappa -bungarotoxin; dNTP, desoxynucleotide triphosphate; TM, transmembrane domain; dn, embryonic day.

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.

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Top
Abstract
Introduction
Procedures
Results
Discussion
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