Neuronal Nicotinic Receptors in the Locust Locusta
migratoria
CLONING AND EXPRESSION*
Bernhard
Hermsen
,
Eva
Stetzer
,
Rüdiger
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
 |
ABSTRACT |
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
subunits, and the other is a
structural
subunit. The existence of at least one more nAChR gene,
probably encoding a
subunit, is indicated.
Based on Northern analysis and in situ hybridization, the
five subunit genes are expressed. loc
1, loc
3, and
loc
1 are the most abundant subunits and are expressed in
similar areas of the head ganglia and retina of the adult locust.
Because Loc
3 binds
-bungarotoxin with high affinity, it may form
a homomeric nAChR subtype such as the mammalian
7 nAChR. Loc
1 and
Loc
1 may then form the predominant heteromeric nAChR in the locust
brain. loc
4 is mainly expressed in optic lobe ganglionic
cells and loc
2 in peripherally located somata of
mushroom body neurons. loc
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 |
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
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
subunits and three
subunits cloned in the rat. Of
these, the
7,
8, and
9 subunits have the unique ability to
form functional homomeric receptors (12-14). Various combinations of
the other
and
subunits also give rise to functional receptors,
as is exemplified by combinations of
4 and
2 subunits and of
3
and
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
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
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
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 |
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 [
-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
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
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 [
-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 Loc
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 Loc
1 (5'-CTCGAGGGACATGTAGAACTCGGAGAGGTC-3') and a mix of two 5'-primers (5'-ACCGCCTCATCAGGCCTGTCACCAACAACTCCGA-3' and
5'-ACCGCCTCATCAGGCCTGTCGGCAACAACTCGGA-3') designed to highly
homologous regions of loc
2 and loc
3
cDNA clones (aa 15-21 of loc
2 and aa 4-10 of
loc
3, respectively). Both 5'-primers contained a silent
mutation to introduce a StuI site (bold letters) which was
later used to fuse the rat
3 nAChR signal peptide to the locust
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 loc
1-specific sequences were isolated by colony
hybridization (Sambrook et al., 1989) using a
[
-32P]dATP-labeled oligo
(5'-CTCGAGGGACATGTAGAACTCGGAGAGGTC-3'), which is highly specific for
loc
1. They were then sequenced, and one of them was fused
to the partial loc
1 cDNA clone.
Construction of Full-length cDNAs--
The
BamHI/EcoRI fragment of the rat
3 nAChR
cDNA (24) and the locust
2,
3, and
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)
(loc
2,
5'-ACCGCCTCATCAGGCCTGTCACCAACAACTCCGA-3'; loc
3,
5'-ACCGCCTCATCAGGCCTGTCGGCAACAACTCGGA-3'; loc
, 5'-ACAAGCTCATCAGGCCTGTGCAGAACATGACGCA-3'). Amplification of
the rat
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
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 loc
1 and loc
2
clones from the "cytoplasmic loop" between TM3 and TM4, for
loc
from parts of the 5'-translated region, for loc
3 from the 3'-untranslated region, and for
loc
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
[
-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 [
-32P]dATP-labeled
probes: a 420-bp HinfI/HinfI fragment of the
loc
2 cDNA, a 230-bp EcoRI/SphI
fragment of the loc
1 cDNA, and a 370-bp EcoRI/SphI fragment of the loc
1
cDNA. Blots were washed with 1× SSC, 65 °C.
Fragments of nAChR
Subunits Obtained by Expression in E. coli
and Binding Studies with
-Bungarotoxin--
cDNA clones were
obtained from the following sources: Drosophila
1 from
Marc Ballivet, Geneva, Switzerland (25); Drosophila
2
from Eckart Gundelfinger, Magdeburg, Germany (26); and
Torpedo
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-
-D-galactopyranoside-induced E. coli transformants (E. coli strain DH5
, 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
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-
BTX
at 4 °C, 30 min, and washing was performed 5 × for 30 min with
1× PBS. 125I-
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 |
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.

View larger version (15K):
[in this window]
[in a new window]
|
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.

View larger version (16K):
[in this window]
[in a new window]
|
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
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.

View larger version (71K):
[in this window]
[in a new window]
|
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
subunits
and one
subunit. The cDNA clones loc
2, loc
3,
and loc
1 encoded for mature subunits, whereas clone
loc
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
subunit (loc
4) but extended only to the end of
transmembrane domain 2. The complete sequence of loc
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 Loc
1-
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 Loc
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
clones, 64% for the
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 Loc
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
-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 Loc
1-3 and Loc
1, respectively) and contains several putative
phosphorylation sites. In the Loc
1 sequence, this region contains a
putative phosphorylation site for cAMP-dependent kinase, as
was also found in non-
subunits from Torpedo (30) and
Drosophila (23).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Hydropathy profile of Loc 2 subunit and
generalized domain structures of L. migratoria nAChR
subunits. The amino acid residues are numbered according to the
Loc 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.

View larger version (25K):
[in this window]
[in a new window]
|
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; D 2/SAD, P17644; ARD,
X04016; SBD, X55676; Mp 1, X81887; Mp 2, X81888; 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
subunit from the locust
Schistocerca gregaria (3). Functional channels were formed
that were gated by micromolar concentrations of nicotine and that were
blocked by
BTX,
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
subunits so far could only be co-expressed in Xenopus
oocytes with the chick neuronal
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
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 loc
clones
the successful co-expression in Xenopus oocytes of
Drosophila
subunit with the vertebrate
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 Loc
3 nAChR Isoform Binds
-Bungarotoxin--
Binding of
the snake neurotoxin
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
subunits were
expressed in E. coli, and the purified fusion proteins were
applied to Western blotting using as probe 125I-labeled
BTX. As is representatively shown in Fig.
6, the Loc
3 fusions (containing aa
126-229 and aa 12-227, respectively) and (as controls) the
Torpedo
fusion (aa 1-246) and Drosophila
1 (ALS) fusion (aa 83-223) bound
BTX, whereas the Loc
2 fusion (aa 1-222) and the Drosophila
2 fusion (aa 3-226) did
not bind I-
BTX. To exclude the possibility of
nonspecific binding of
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
BTX to Loc
3 (aa 12-290),
Torpedo
1 (aa 143-201), rat
1 (aa 1-210), and rat
7 (aa 80-213) was demonstrated. Selective binding of
BTX by
Loc
3, as compared with Loc
2, cannot be explained by differences
in sequence in the region around the two adjacent cysteines of
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
BTX is discontinuously distributed within the N-terminal
region of the
subunit has previously been reported for nAChRs from
other species (34).

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 6.
Binding of -bungarotoxin to fusion
proteins containing fragments of the N-terminal extracellular region of
nAChR subunits from several species. Samples of affinity
purified fusion proteins containing nAChR fragments from
Torpedo (aa 1-246, lane 1),
Drosophila 2 (aa 3-226, lane 2), Loc 3 (aa
126-229, lane 3), Loc 3 (aa 12-227, lane 4),
Loc 2 (aa 1-222, lane 5), and Drosophila 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- 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 BTX was detected for Torpedo- , Loc 3, and
D 1.
|
|
From its
BTX binding properties, Loc
3 is a candidate for a
homo-oligomeric nAChR, such as the
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 Loc
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
BTX) for binding to the Torpedo nAChR (34, 38, 39). Selective binding of WF6 was observed only to the
Loc
3 fusion proteins and (as controls) to those from Torpedo
and D
1 (data not shown). These observations
agree with previous findings in that the attachment point patterns
within the binding sites for
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 loc
1,
loc
2, and loc
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
cDNA probe always showed two transcripts (6.1 and 4.1 kilobase pairs), suggesting cross-hybridization with a second (as yet
unidentified)
subunit mRNA.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 7.
Developmental stage-specific expression of
L. migratoria nAChR subunits 1, 2, and 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 loc 1, loc 2, and loc 1.
A, 420-bp long HinfI/HinfI fragment
from loc 2; B, 370-bp long
EcoRI/SphI fragment from loc 1;
C, 230-bp long EcoRI/SphI fragment
from loc 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).

View larger version (174K):
[in this window]
[in a new window]
|
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 1
(B), 2 (C), 3 (D), 4
(E), and 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 1, 2, 3, and 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).
|
|

View larger version (94K):
[in this window]
[in a new window]
|
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 1
(C), 3 (D), 4 (F and
G), and 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 1, 3, and
1 was detected in primary and secondary pigment cells of single
ommatidia in the retina. In contrast, 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, 4 mRNA was
located in the perinuclear region of ganglionic cell bodies.
|
|
As shown in Fig. 8, B-F, mRNA encoding
loc
1-3 and loc
1, but not
loc
4, was detected in the mushroom bodies of the adult locust. The strongest expression was observed for loc
1
mRNA, in the cytosol of neurons whose cell bodies are located in
the center of the mushroom bodies. In roughly the same area,
loc
3 and loc
1 mRNA was detected.
loc
3 mRNA was expressed to a much lower extent than
loc
1 or loc
1. loc
4 mRNA
was not detected in mushroom bodies. loc
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
loc
1, loc
3, and loc
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, loc
1 mRNA was most abundant, followed by loc
1 and loc
3 mRNA.
loc
2 and loc
4 mRNA were absent. Transcripts of the three subunits were located in primary and secondary
pigment cells which surround the crystal cones. Whereas loc
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, Loc
1, Loc
3 and Loc
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 loc
1
mRNA was abundantly expressed in the protocerebral lobe, the
developing mushroom body (Fig. 10F), the
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. loc
1 mRNA was also detected in the ectoderm
anlage from which the eye develops (Fig. 10F).

View larger version (142K):
[in this window]
[in a new window]
|
Fig. 10.
Expression of nAChR isoforms in the brain
and periphery of a d8 embryo. A, overview of an in
situ hybridization with a 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 1 (B), 2
(C), 3 (D and G), 4
(E), and 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 1 mRNA
(F) but no mRNA coding for isoforms was expressed,
suggesting that expression of the structural subunit may precede
formation of functional channels of and subunits.
|
|
Outside of the head area, loc
3 mRNA was mainly
detected in isolated cells interspersed in the somatogastric epithelium
(Fig. 10G). Additional limited expression of
loc
3 mRNA was observed in peripheral ganglion cells
distributed in the mesenchyme below the somatogastric epithelium. In
contrast, loc
1 mRNA 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 course of this
study, we have identified in the locust L. migratoria six
nAChR subunit genes. Three cDNAs encoding
subunits and one cDNA encoding a
subunit were obtained as full-length clones. In
addition we have isolated a partial nAChR
subunit cDNA and have
identified in Northern blots (Fig. 3) a sixth mRNA which probably
encodes another
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. Loc
1, Loc
3, and Loc
1 are the isoforms most
abundantly expressed in the head ganglia and the retina, whereas
Loc
4 is mainly expressed in optic lobe ganglionic cells. Based on
its
BTX binding properties (Fig. 6), Loc
3 is a candidate for a
homomeric nAChR. Extending this line of arguments, Loc
1 and Loc
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
Loc
1, Loc
3, and Loc
1 together form a heteromeric nAChR
(42-45). The early presence of Loc
1 in the embryo of the locust
suggests that its expression may be a prerequisite for functional
assembly of some hetero-oligomeric nAChR subtypes. loc
2
and loc
4 mRNA are much less abundant and have distinctly different expression patterns, suggesting that they do
not form subunits of nAChR subtypes containing
1,
3, and
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) Loc
1 and MARA1 (and the
1 subunit of the lepidopteran Heliothis virescens which
was recently cloned in our
laboratory)2; 2) Loc
2 and
SBD; 3) Loc
3, ALS, and Mp
2; 4) Loc
1 and ARD; and 5) SAD and
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
3 nAChR expressed in
skin keratinocytes has been reported to regulate cell adhesion and
motility. The expression of Loc
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
and non-
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
and one
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
clone. We thank Marc Ballivet (Geneva) for
providing the rat
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;
BTX,
-bungarotoxin;
BTX,
-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.
 |
REFERENCES |
-
Gerschenfeld, H. M.
(1973)
Physiol. Rev.
53,
1-119[Free Full Text]
-
Gundelfinger, E. D.,
and Hess, N.
(1992)
Biochim. Biophys. Acta
1137,
299-308[Medline]
[Order article via Infotrieve]
-
Marshall, J.,
Buckingham, S. D.,
Shingai, R.,
Lunt, G. G.,
Goosey, M. W.,
Darlison, M. G.,
Satelle, D. B.,
and Barnard, E. A.
(1990)
EMBO J.
9,
4391-4398[Abstract]
-
Eastham, H. M.,
Lind, R. J.,
Clarke, B. S.,
Towner, P.,
Reynolds, S. E.,
Wolstenholme, A. J.,
and Wonnacott, S.
(1998)
Eur. J. Neurosci.
10,
879-889[CrossRef][Medline]
[Order article via Infotrieve]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual, Vol. 1, pp. 7.14-7.15, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
-
Satelle, D. B.
(1980)
Adv. Insect Physiol.
15,
215-315
-
Benke, D.,
and Breer, H.
(1989)
Comp. Biochem. Physiol.
1,
71-80
-
Breer, H.,
and Satelle, D. B.
(1987)
J. Insect Physiol.
33,
771-790[CrossRef]
-
Benson, J. A.
(1993)
in
Comparative Molecular Neurobiology (Pichon, Y., ed), pp. 390-413, Birkhäuser Verlag, Basel
-
Breer, H.
(1981)
Neurochem. Int.
3,
43-52
-
Lindstrom, J.
(1996)
Ion Channels
4,
377-450[Medline]
[Order article via Infotrieve]
-
Bertrand, D.,
Bertrand, S.,
and Ballivet, M.
(1992)
Neurosci. Lett.
146,
87-90[CrossRef][Medline]
[Order article via Infotrieve]
-
Gerzanich, V.,
Anand, R.,
and Lindstrom, J.
(1994)
Mol. Pharmacol.
45,
212-220[Abstract]
-
Elgoyhen, A. B.,
Johnson, D. S.,
Boulter, J.,
Vetter, D. E.,
and Heinemann, S.
(1994)
Cell
18,
705-715
-
Alkondon, M.,
Reinhardt, S.,
Lobron, C.,
Hermsen, B.,
Maelicke, A.,
and Albuquerque, E. X.
(1994)
J. Pharmacol. Exp. Ther.
271,
494-506[Abstract]
-
Le Novère, N.,
and Changeux, J. P.
(1995)
J. Mol. Evol.
40,
155-172[Medline]
[Order article via Infotrieve]
-
Breer, H.,
Kleene, R.,
and Hinz, G.
(1985)
J. Neurosci.
5,
3386-3392[Abstract]
-
Hanke, W.,
and Breer, H.
(1986)
Nature
321,
171-174[Medline]
[Order article via Infotrieve]
-
Lansdell, S. J.,
Schmitt, B.,
Betz, H.,
Satelle, D. B.,
and Millar, N.
(1997)
J. Neurochem.
68,
1812-1819[Medline]
[Order article via Infotrieve]
-
Satelle, D. B.,
Buckingham, S. D.,
Wafford, K. A.,
Sherby, S. M.,
Bakry, N. M.,
Eldefrawi, A. T.,
Eldefrawi, M. E.,
and May, T. E.
(1989)
Proc. R. Soc. Lond. B Biol. Sci.
237,
501-514[Medline]
[Order article via Infotrieve]
-
Zwart, R.,
Oortgiesen, M.,
and Vijverberg, H. P. M.
(1994)
Pestic. Biochem. Physiol.
48,
202-213[CrossRef]
-
Davis, L. G.,
Dibner, M. D.,
and Battey, J. F.
(1986)
Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., New York
-
Hermans-Borgmeyer, I.,
Zopf, D.,
Ryseck, R.-P.,
Hovemann, B.,
Betz, H.,
and Gundelfinger, E. D.
(1986)
EMBO J.
5,
1503-1508
-
Deneris, E. S.,
Conolly, J.,
Rogers, S. W.,
and Duvoisin, R.
(1991)
Trends Pharmacol. Sci.
12,
34-30[Medline]
[Order article via Infotrieve]
-
Bossy, B.,
Ballivet, M.,
and Spierer, P.
(1988)
EMBO J.
7,
611-618[Abstract]
-
Baumann, A.,
Jonas, P.,
and Gundelfinger, E. D.
(1990)
Nucleic Acids Res.
18,
3640[Medline]
[Order article via Infotrieve]
-
Usherwood, P. N. R.,
Giles, D.,
and Suter, C.
(1980)
Insect Neurobiology and Pesticide Action (Neurotox `79), pp. 115-128, Society of Chemical Industry, London
-
Fenwick, E.,
Marty, A.,
and Neher, E.
(1982)
J. Physiol. (Lond.)
331,
577-597[Abstract]
-
Benson, J. A.
(1988)
in
Nato ASI Series H (Clementi, F., Gotti, C., and Sher, E., eds), Vol. 25, pp. 227-240, Springer-Verlag, Berlin
-
Safran, A.,
Provenzano, C.,
Sagi-Eisenberg, R.,
and Fuchs, S.
(1989)
in
NATO ASI Series, Series H: Cell Biology (Maelicke, A., ed), Vol. 32, pp. 373-380, Springer-Verlag, Berlin
-
Bertrand, D.,
Ballivet, M.,
Gomez, M.,
Bertrand, S.,
Phannavong, B.,
and Gundelfinger, E. D
(1994)
Eur. J. Neurosci.
6,
869-875[Medline]
[Order article via Infotrieve]
-
Gehle, V. M.,
and Sumikawa, K.
(1991)
Mol. Brain Res.
11,
17-25[Medline]
[Order article via Infotrieve]
-
Buller, A. L.,
and White, M. M.
(1990)
J. Membr. Biol.
115,
179-189[Medline]
[Order article via Infotrieve]
-
Conti-Tronconi, B. M.,
Tang, F.,
Diethelm, B. M.,
Spencer, S. R.,
Reinhardt-Maelicke, S.,
and Maelicke, A.
(1990)
Biochemistry
29,
6221-6230[Medline]
[Order article via Infotrieve]
-
Alkondon, M.,
and Albuquerque, E. X.
(1993)
J. Pharmacol. Exp. Ther.
265,
1455-1473[Abstract]
-
Couturier, J.,
Erkman, C.,
Valera, S.,
Rungger, D.,
Bertrand, S.,
Boulter, J.,
Ballivet, M.,
and Bertrand, D.
(1990)
J. Biol. Chem.
265,
17560-17567[Abstract/Free Full Text]
-
Albuquerque, E. X.,
Pereira, E. F.,
Alkondon, M.,
Schrattenholz, A.,
and Maelicke, A.
(1997)
J. Recept. Signal Transduct. Res.
17,
243-266[Medline]
[Order article via Infotrieve]
-
Fels, G.,
Plümer-Wilk, R.,
Schreiber, M.,
and Maelicke, A.
(1986)
J. Biol. Chem.
261,
15746-15754[Abstract/Free Full Text]
-
Schröder, B.,
Reinhardt-Maelicke, S.,
Schrattenholz, A.,
McLane, K. E.,
Kretschmer, A.,
Conti-Tronconi, B. M.,
and Maelicke, A.
(1994)
J. Biol. Chem.
269,
10407-10416[Abstract/Free Full Text]
-
Conti-Tronconi, B. M.,
Diethelm, B. M.,
Wu, X.,
Tang, F.,
Bertazzon, T.,
Schröder, B.,
Reinhardt-Maelicke, S.,
and Maelicke, A.
(1991)
Biochemistry
30,
2575-2584[Medline]
[Order article via Infotrieve]
-
Leitch, B.,
Watkins, B. L.,
and Burrows, M.
(1993)
J. Comp. Neurol.
334,
47-58[Medline]
[Order article via Infotrieve]
-
Conroy, W.,
and Berg, D.
(1995)
J. Biol. Chem.
270,
4424-4431[Abstract/Free Full Text]
-
Wang, F.,
Gerzanich, V.,
Wells, G.,
Anand, R.,
Peng, X.,
Keyser, K.,
and Lindstrom, J.
(1996)
J. Biol. Chem.
271,
17656-17665[Abstract/Free Full Text]
-
Raminez-Torre, J.,
Yu, C.,
Qu, X.,
Perin, F.,
Karlin, A.,
and Role, L.
(1996)
Nature
380,
347-351[CrossRef][Medline]
[Order article via Infotrieve]
-
Forsayeth, J.,
and Kobrin, E.
(1997)
J. Neurosci.
17,
1531-1538[Abstract/Free Full Text]
-
Stetzer, E.,
Ebbinghaus, U.,
Storch, A.,
Poteur, L.,
Schrattenholz, A.,
Kramer, G.,
Methfessel, C.,
and Maelicke, A.
(1996)
FEBS Lett.
397,
39-44[CrossRef][Medline]
[Order article via Infotrieve]
-
Grando, S. A.,
Horton, R. M.,
Pereira, E. F. R.,
George, P. M.,
Diethelm-Okita, B. M.,
Albuquerque, E. X.,
and Conti-Fine, B. M.
(1995)
J. Invest. Dermatol.
105,
774-781[Abstract]
-
Methfessel, C.,
Witzemann, V.,
Takahashi, T.,
Mishina, M.,
Numa, S.,
and Sakmann, B.
(1986)
Pfluegers Arch.
407,
577-588[Medline]
[Order article via Infotrieve]
-
Bertrand, D.,
Galzi, J.,
Devilliers-Thiery, A.,
Bertrand, S.,
and Changeux, J. P.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
6971-6975[Abstract]
-
Maelicke, A.
(1984)
Angew. Chem. Int. Ed.
23,
195-221
-
Weber, H.
(1938)
Compendium of Entomology, Fischer Verlag, Jena, Germany
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.