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Vol. 53, Issue 6, 1112-1119, June 1998
3 Subunit
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101
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Summary |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Neuronal bungarotoxin (NBT) is a highly selective, slowly reversible,
competitive antagonist of the 
3
2 neuronal nicotinic receptor.
Contributions to NBT sensitivity are made by both the 3 and 2
subunits. We used a chimeric subunit to demonstrate that the entire
3 contribution lies within sequence segment 84-215. Construction
and analysis of a series of mutant 3 subunits identified seven amino
acid residues (Thr143, Tyr184, Lys185, His186, Ile188, Gln198, Ser203)
within this region that contribute to NBT sensitivity. Changing Thr143
to lysine, as in 2, resulted in a ~1000-fold loss of NBT
sensitivity. The effect on NBT sensitivity of changing each of the
other six residues ranged from 1.8- to 40.5-fold. More extensive
mutagenesis demonstrated that Thr143 serves as part of the consensus
sequence for glycosylation at N141, and it is this glycosylation that
is the determinant of NBT sensitivity. Only serine could substitute for
threonine to maintain full NBT sensitivity, and changing Asn141 to
alanine resulted in a ~300-fold loss of NBT sensitivity. The chimera
2-181-3, containing all identified determinants except the
glycosylation site, formed receptors insensitive to 300 nM
NBT. Installation of threonine to complete the glycosylation consensus
site in this chimera conferred NBT sensitivity only 10-fold less than
that of wild-type 32. These seven determinants of NBT sensitivity
are located in close proximity to a series of conserved residues that
are common features of all nicotinic receptor binding sites.
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Introduction |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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nAChRs
are found throughout the central and
peripheral nervous systems. Although nAChRs are responsible for rapid
neurotransmission in the periphery, these receptors seem to play a
modulatory role in the central nervous system (Gray et al.,
1996
; Role and Berg, 1996). Neuronal nAChRs form in a manner similar to
muscle nAChRs, as a pentameric assembly of subunits (Anand et
al., 1991; Cooper et al., 1991). These receptors
assemble from various combinations of 
11 distinct subunits, 2-9
and 2-4 (Sargent, 1993; Elgoyhen et al., 1994). The
resulting nAChRs differ pharmacologically and biophysically, depending
on subunit composition (Role, 1992).
The ligand binding sites of muscle type nAChRs are formed at the
interface between and non- (
and 
) subunits (Karlin and
Akabas, 1995). The ligand binding sites of neuronal nAChRs seem to be
formed in a similar fashion; both and subunits contribute to
the pharmacological properties of these receptors (Duvoisin et
al., 1989; Luetje and Patrick, 1991). Affinity labeling techniques
have identified amino acid residues on the , , and subunits
of muscle-type nAChRs that contribute to the structure of the ligand
binding sites (Karlin and Akabas, 1995). These residues are highly
conserved among neuronal nAChR subunits and thus are features of
nicotinic binding sites common to all nAChRs. These conserved residues
cannot account for the pharmacological diversity that exists among
neuronal nAChRs. These differences must be due to residues that differ
among neuronal nAChR subunits. As an approach to the identification of
residues responsible for conferring pharmacological differences, we
constructed and analyzed chimeras of pharmacologically distinct
subunits and then used site-directed mutagenesis to identify residues
that confer specific pharmacological features on neuronal nAChRs
(Luetje et al., 1993; Harvey et al., 1996, 1997;
Harvey and Luetje, 1996).
NBT, a dimeric protein toxin formed by 66 residue monomers, is isolated
from the venom of Bungarus multicinctus. NBT is a highly
selective, slowly reversible, competitive antagonist of the 32
neuronal nAChR subunit combination. Changing either the or subunit results in a dramatic loss in NBT sensitivity (Duvoisin et al., 1989; Luetje et al., 1990). This high
degree of specificity makes NBT an ideal probe for studying the ligand
binding sites of neuronal nAChRs. We previously used NBT to identify
Thr59 of the 2 subunit as a critical determinant of competitive
antagonist sensitivity (Harvey and Luetje, 1996). We also used NBT to
identify three distinct sequence segments on the 3 subunit (84-121,
121-181, 195-215) that contain determinants of competitive antagonist
sensitivity (Luetje et al., 1993). Each of these regions was
found to be necessary for full NBT sensitivity. The 121-181 and
195-215 regions were particularly critical because chimeric subunits
lacking either of these regions formed receptors that were insensitive
to high concentrations of NBT. We now show that residues lying within segment 84-215 of 3 are entirely sufficient to confer full NBT sensitivity on the 2 subunit. We also use site-directed mutagenesis to identify seven residues within this region that contribute to NBT
sensitivity. One of these residues, Thr143, fulfills its role as a
determinant by serving as part of the consensus sequence for
glycosylation at Asn141.
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Experimental Procedures |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Materials. Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). The care and use of X. laevis frogs in this study were approved by the University of Miami Animal Research Committee and meet the guidelines of the National Institutes of Health. RNA transcription kits were from Ambion (Austin, TX). ACh, atropine, and 3-aminobenzoic acid ethyl ester were from Sigma Chemical (St. Louis, MO). Collagenase B was from Boehringer-Mannheim (Indianapolis, IN). Sequenase 2.0 kits were from Amersham Life Sciences (Cleveland, OH). NBT was from Biotoxins (St. Cloud, FL). CloneAmp kits were from GIBCO BRL (Gaithersburg, MD).
Mutagenesis and construction of chimeric receptors.
Chimeric
and mutant subunits were constructed using PCR (Higuchi, 1990). Our
notation for mutant subunits is to list the naturally occurring residue
followed by the position of that residue and then followed by the
change that has been made. For example, the mutant subunit 3, I188K
is an 3 subunit in which Ile188 has been changed to a lysine. PCR
products were subcloned into the pAMP1 vector using a CloneAmp kit. To
minimize the amount of PCR product in the final construct that would
have to be sequenced, as much PCR product as possible was replaced with
appropriate wild-type sequence using existing restriction sites.
Remaining sequence derived from PCR product was confirmed by sequencing using Sequenase 2.0.
Injection of in vitro synthesized RNA into
X. laevis oocytes.
m7G(5')ppp(5')G capped cRNA was synthesized
in vitro from linearized template DNA encoding the 2,
3, 4, and 2 subunits, as well as the various chimeric and
mutant subunits, using an Ambion mMessage mMachine kit. Mature
X. laevis frogs were anesthetized by submersion
in 0.1% 3-aminobenzoic acid ethyl ester, and oocytes were surgically
removed. Follicle cells were removed by treatment with collagenase B
for 2 hr at room temperature. Each oocyte was injected with 10-30 ng
of cRNA in 50 nl of water and incubated at 19° in modified Barth's
saline (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM
CaNO3, 0.41 mM
CaCl2, 0.82 mM
MgSO4, 100 µg/ml gentamicin, 15 mM
HEPES, pH 7.6) for 2-7 days. RNA transcripts encoding each subunit
were injected into oocytes at a molar ratio of 1:1.
Electrophysiological recordings.
Oocytes were perfused at
room temperature (20-25°) in a 300-µl chamber with perfusion
solution (115 mM NaCl, 1.8 mM
CaCl2, 2.5 mM KCl, 10 mM
HEPES, pH 7.2, 1.0 µM atropine). Perfusion was continuous
at a rate of ~20 ml/min. ACh was diluted in perfusion solution, and
the oocytes were exposed to ACh for ~10 sec, using a solenoid valve.
NBT sensitivity was tested by comparing ACh-induced current responses
before and after the oocytes were incubated for 30 min in perfusion
solution containing various concentrations of NBT and 100 µg/ml
bovine serum albumin. We have shown previously that bovine serum
albumin alone has no effect (Luetje et al., 1990). A 10-sec
wash was included before the postincubation ACh exposure. The
postincubation ACh response is presented as a percentage of the
preincubation ACh response. ACh concentrations were below the
EC50 value for each receptor to avoid extensive
desensitization. Preincubation with NBT results in a slowly reversible
competitive blockade of 32 but not 22 (Luetje et
al., 1990). Coapplication of NBT with agonist reveals a rapidly
reversible blockade of subunit combinations other than 32. Our
experimental protocol incorporates a wash period after NBT incubation
and before measurement of the postincubation ACh response. This wash
step eliminates the rapidly reversible blockade of subunit combinations
other than 32. Thus, our protocol detects only slowly reversible
blockade by NBT. The slowly reversible nature of NBT blockade allows
the postincubation ACh response to be measured without coapplication of
toxin. Because toxin and ACh were not in direct competition, the degree
of observed block was not dependent on the concentration of ACh, and
the ACh concentration used for each receptor did not have to be
equipotent with the ACh concentrations used for other receptors.
70 mV, using a
TEV-200 voltage-clamp unit (Dagan, Minneapolis, MN). Micropipettes were
filled with 3 M KCl and had resistances of 0.5-1.0
M
. Agonist-induced responses were captured, stored, and
analyzed on a Macintosh IIci computer using a data acquisition program
written with LabVIEW (National Instruments, Austin, TX) and LIBI
(University of Arizona, Tuscon, AZ) software (Luetje and Patrick,
1991).
Fold differences between NBT dose-inhibition data for various receptors
were estimated through visual inspection of Figs. 2, 5, and 6.
Comparisons were made at 50% inhibition unless otherwise noted.
Statistical significance was determined by using a two-sample t test.
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Results |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Residues 84-215 of the 3 subunit are sufficient to confer NBT
sensitivity on the 2 subunit.
We previously demonstrated that
three-sequence segments of the 3 subunit are important for NBT
sensitivity (Luetje et al., 1993). In that study, a series
of chimeric subunits were constructed that consisted of portions of
the 3 and 2 subunits. Each of the three regions identified
(84-121, 121-181, and 195-215) was found to be necessary, but not
sufficient, to achieve the full NBT sensitivity of the 32
receptor. No data were obtained regarding the potential involvement of
the 181-195 region. Residues 1-83 and residues from 216 to the
carboxyl terminus were found to be unnecessary for NBT sensitivity.
3 are
sufficient to confer complete NBT sensitivity, we constructed the chimeric subunit 2-84-3-215-2. This chimera consists of
the 2 subunit with residues 84-215 replaced by 3 sequence. On
expression in X. laevis oocytes, in combination with the
2 subunit, this chimera forms receptors that are sensitive to
blockade by NBT. In Fig. 1A, we show ACh
induced current responses obtained from oocytes expressing 32,
22, or 2-84-3-215-2 2 before and after a 30-min
incubation with 10 nM NBT. The 32 receptors are antagonized by 10 nM NBT. In contrast, the 22
receptors are insensitive to this concentration of toxin. In fact,
concentrations of NBT as high as 1 µM do not cause
blockade of the 22 receptor (Luetje et al., 1993). The
2-84-3-215-2 2 receptors display a sensitivity to 10 nM NBT similar to that of 32. In Fig. 1B, we compared
the sensitivity of 2-84-3-215-2 2 and 32 to a range
of NBT concentrations and found the NBT sensitivity of these two
receptors to be indistinguishable. This result confirms the importance
of residues within the 84-215 segment and rules out any requirement
for residues between the amino terminus and position 83 or for residues
between position 216 and the carboxyl terminus.
|
; Wolf
et al., 1988; Loring et al., 1989), we wanted to
confirm that NBT blockade of the rat 32 subunit combination
expressed in oocytes also was competitive. We previously demonstrated
that DHE is a competitive antagonist of the 32 subunit
combination (Harvey and Luetje, 1996). We took advantage of the
difference in the rate of recovery of 32 receptors from DHE
and NBT blockade to demonstrate that DHE competes with NBT for
binding to 32 receptors (Fig. 2). A
2.5-min wash period was included after the standard 30-min incubation
with NBT. Under these conditions, NBT alone antagonized 32 with
an IC50 value of 2.9 nM. In contrast, 30-min incubation with 100 nM DHE followed by a 2.5-min
wash period resulted in no blockade of 32. This lack of blockade is due to the rapid off-rate of DHE from the receptor. When 100 nM DHE is included in NBT incubations, the
IC50 value for receptor blockade was shifted
6.6-fold to 19 nM. This rightward shift of the NBT
concentration-inhibition relationship in the presence of DHE
demonstrates direct competition between NBT and DHE.
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Identification of seven residues (Thr143, Tyr184, Lys185, His186,
Ile188, Gln198, and Ser203) on 3 that contribute to NBT
sensitivity.
The 3 and 2 subunits differ at 41 of the 132 residues in segment 84-215 (Fig. 3). To
identify the amino acid residues in this region that are responsible
for NBT sensitivity, we constructed a series of mutant 3 subunits.
Residues in 3 were changed to what occurs at the analogous position
in 2. In a few cases, we changed two or three residues
simultaneously. Each mutant subunit then was coexpressed with the 2
subunit and tested for sensitivity to blockade of ACh-induced current
responses by 10 nM NBT (Fig. 4). We found that mutations at seven
positions resulted in a significant loss in NBT sensitivity.
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|
). However, mutation analysis in
Fig. 4 did not reveal any critical residues. The most likely explanation is that several of the residues in this region each make
minor contributions that are too small to be detected individually in
our assay. Within region 121-181, the T143K mutation caused a
significant loss of NBT sensitivity. This residue is of particular interest because it is located directly adjacent to Cys142, one of two
cysteines in nicotinic receptor subunits that form the conserved
cysteine-loop structure. Our analysis of the role of Thr143 in
determining NBT sensitivity is presented below.
In previous work (Luetje et al., 1993), it was unclear
whether amino acid residues between 181 and 195 were important for NBT
sensitivity. However, it is clear in Fig. 4 that this region is
critical. Four of the seven amino acid residues that we identified on
the 3 subunit lie within this region (Tyr184, Lys185, His186, Ile188). In Fig. 5A, we examined the
effect of changing each of these residues on sensitivity to a range of
NBT concentrations. The change at position 184 from a tyrosine to a
threonine, a loss of an aromatic ring, had a modest (1.8-fold) effect
on NBT sensitivity. A similarly modest (2.4-fold) effect occurred when
His186 was changed to an asparagine. A larger (11.2-fold) effect was
achieved by changing Ile188 to lysine, going from a hydrophobic residue to a positively charged residue. Mutation of Lys185 to tyrosine, resulting in a loss of the positive charge and gain of an aromatic ring, resulted in a large shift in NBT sensitivity (40.5-fold).
|
3 to proline, as in 2, causes a 9.5-fold loss
in NBT sensitivity. The S203Y mutation, a gain of an aromatic ring,
causes a 5.9-fold loss in NBT sensitivity.
The 4 subunit has a threonine at position 143 (and an asparagine at
141; see below), similar to 3, but lacks the other six residues that
we identified as determinants of NBT sensitivity in regions 181-195
and 195-215 (see Fig. 3). As might be expected, 42 receptors
display little sensitivity to NBT. Even at a concentration of 1 µM, NBT is able to block 42 by only 15.9 ± 1.4% (five oocytes). We hypothesized that installation of an
additional determinant might increase the NBT sensitivity of 42.
In previous work, we found that changing P198 of the chimera
3-195-2 to glutamine increased the NBT sensitivity of the
resulting receptors (Luetje et al., 1993). Thus, we sought
to test the role of glutamine at position 198 by changing Pro198 of
4 to glutamine, as in 3. We found that the mutant subunit (4,
P198Q) formed receptors with a significantly increased NBT sensitivity.
At a concentration of 1 µM, NBT was able to block 4,
P198Q 2 by 77.4 ± 6.8% (four oocytes; significantly different
from 42, p < 0.001).
Thr143 contributes to NBT sensitivity as part of a glycosylation
consensus sequence.
Receptors formed by 3, T143K are
insensitive to both 10 and 100 nM NBT; however, 300 nM NBT is able to cause partial blockade (Fig.
6B). The degree of blockade of 3,
T143K 2 receptors by 300 nM NBT is not significantly
different from the blockade of 32 receptors achieved by 300 pM NBT. Thus, the loss in NBT sensitivity due to the T143K
mutation is ~1000-fold. In contrast to the large effect on NBT
sensitivity, the T143K mutation has little effect on ACh sensitivity.
The ACh EC50 value of 3, T143K 2 is
30.3 ± 8.5 µM, similar to the
EC50 value of 70.8 ± 19.6 µM
of 32 (Harvey and Luetje, 1996).
|
3 and 2, but only
3 has the appropriate residue at position 143 that would allow
glycosylation. To test this possibility, we changed Asn141 to alanine,
thus precluding the possibility of glycosylation at position 141. Similar to what we observed for the T143K mutation, the N141A mutation
has only a minimal effect on ACh sensitivity
(EC50 = 18.2 ± 10.9 µM).
However, the N141A mutation has a large effect on NBT sensitivity. In
Fig. 6B, we show that receptors formed by the N141A mutant are
~300-fold less sensitive to NBT than are 32 receptors. This
result suggests that glycosylation at position 141 of the 3 subunit
is an essential requirement for NBT sensitivity.
A better test of the role of glycosylation at N141 would be to install
this glycosylation site in an insensitive subunit. As discussed above,
the 2 subunit has an asparagine at position 146 (analogous to Asn141
of 3) but lacks the appropriate threonine or serine at position 148. Thus, installation of a threonine at this site would complete the
consensus for glycosylation. However, our previous work with chimeric
subunits (Luetje et al., 1993) suggests that simply
completing the glycosylation site in 2 would have little effect on
NBT sensitivity. The 3-195-2 chimera, which possesses most of
the determinants that we have identified in this study (the
glycosylation site, as well as Tyr184, Lys185, His186, and Ile188), was
insensitive to 1 µM NBT. Thus, to test the effect of
adding the glycosylation site, many of the other critical residues will
have to be present. To achieve this, we decided to test the effect of
completing the glycosylation site on the chimeric subunit
2-181-3. This subunit possesses all critical residues that we
have identified, with the exception of the glycosylation site. As shown
in Fig. 6C, receptors formed by this chimera are insensitive to 300 nM NBT. Completion of the glycosylation site in this
chimera, yielding 2(K148T)-181-3, resulted in a dramatic increase
in NBT sensitivity. Receptors formed by this mutant chimera were
sensitive to NBT with an IC50 value of ~13
nM. This is only 10-fold less than the sensitivity of
receptors formed by wild-type 3. Taken together, the results presented in Fig. 6 strongly suggest that glycosylation of Asn141 of
3 is critical for achieving full NBT sensitivity.
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Discussion |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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We identified seven amino acid residues on the 3 subunit of
neuronal nicotinic receptors that contribute to NBT sensitivity. These
residues lie near a series of conserved residues, thought to comprise
the common features of all nAChR binding sites (Fig. 7). Originally identified on the
Torpedo californica electric organ nAChR subunit using affinity labeling techniques (Karlin and Akabas, 1995),
these residues are highly conserved among nAChR subunits, including
the rat neuronal 2 and 3 subunits. The fact that these residues
are common to both 2 and 3 excludes them from responsibility for
the different pharmacological properties displayed by receptors formed
by these subunits. We show that a series of residues positioned near
these common residues are responsible for the differing specificity of
these receptors. The glycosylation consensus consisting of Asn141 and
Thr143 lies close to the conserved Trp149, whereas a cluster of common
determinants (Tyr190, Cys192, Cys193, Tyr197) is flanked by Tyr184,
Lys185, His186, and Ile188 on the amino-terminal side and by Gln198 and Ser203 on the carboxyl-terminal side. Interestingly, scanning cysteine
accessibility mutagenesis has demonstrated the proximity of His186 and
Ile188 of the mouse muscle subunit to the ACh binding site
(McLaughlin et al., 1995).
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The large size of NBT (a dimer of 66 residue monomers) is a potential
problem when using this toxin to study the ligand binding site.
Although NBT is a competitive antagonist (Halvorsen and Berg, 1987;
Wolf et al., 1988; Loring et al., 1989; current
study), some interactions between NBT and the receptor could be quite distant from the binding site. Thus, it is encouraging that we find
overlap in our mapping of determinants of moderately sized (-CTx-MII) and small (DHE) competitive antagonists on both and subunits. Two of the residues on 3 that we have identified as NBT determinants (Lys185 and Ile188) also serve as determinants of
-CTx-MII sensitivity (Harvey et al., 1997). Sequence
segment 195-215 of 3, which contains Gln198 and Ser203, also
contains a determinant of DHE sensitivity, although we have not yet
identified the individual residue or residues involved (Harvey et
al., 1996). The situation is even more striking for the 2
subunit. We found that a major determinant of NBT sensitivity, T59,
also is a determinant of both DHE and -CTx-MII sensitivity
(Harvey and Luetje, 1996; Harvey et al., 1997). This overlap
in the determinant maps for competitive antagonist sensitivity confirms
NBT as a useful tool to investigate the structure of the ligand binding
sites of neuronal nAChRs.
A different approach to the identification of residues responsible for
NBT sensitivity has been to test a series of peptides, corresponding to
the 3 sequence, for the ability to bind
125I-NBT (McLane et al., 1990, 1991,
1993). Peptides representing residues 1-18 and 51-70 of 3 were
shown to bind 125I-NBT with low affinity, whereas
the 2 peptide analogous to 3:51-70 did not bind NBT. In Fig. 1,
we demonstrate that residues 1-83 of 3, which includes both 3
peptides, are unnecessary for NBT blockade of the 32 receptor.
McLane et al. (1990) also showed that the 3 peptides
180-199 and 183-201, although not able to bind
125I-NBT in a solid-phase assay, were able to
partially inhibit the binding of 125I-NBT to PC12
cells. Some of the residues that we identified as determinants of NBT
sensitivity (Tyr184, Lys185, His186, Ile188, Gln198) lie within the
region covered by these two peptides. Thus, although studies using
peptides may identify sequence segments important for ligand binding
(i.e., 3:180-199 and 3:183-201), the relative affinities of
different peptides for ligands may not be relevant to the role those
sequences play in the intact receptor.
Our work strongly suggests that Thr143 fulfills its role as a
determinant of NBT sensitivity by serving as part of a glycosylation consensus sequence for glycosylation at Asn141. It is the presence of
glycosylation at Asn141 that seems to be required for NBT blockade of
3-containing nAChRs. This surprising result contrasts sharply with
the role of glycosylation in conferring -bungarotoxin resistance on
nAChRs of snake and mongoose muscle, where the presence of an
additional glycosylation confers resistance, presumably through steric
hindrance (Kreienkamp et al., 1994; Keller et
al., 1995).
The presence of the consensus for glycosylation at Asn141 is necessary,
but not sufficient, to confer NBT sensitivity. This site is conserved
among most nicotinic receptor subunits, even those that form receptors
insensitive to NBT. These subunits most likely lack other critical
determinants of NBT sensitivity. This dependence on additional residues
for achieving NBT sensitivity is demonstrated in our previous work
(Luetje et al., 1993). We found that the chimera
3-195-2, which possesses the glycosylation site and lacks only
the determinants between 195 and 215, formed receptors that were
insensitive to NBT concentrations as high as 1 µM.
Thr143 and Asn141 lie on either side of Cys142, which forms a disulfide
bond with Cys128, raising the question of whether glycosylation can
occur at Asn141. In fact, the analogous site on T. californica electric organ subunit has been demonstrated to be
glycosylated (Strecker et al., 1994). Rickert and Imperiali (1995) have shown, using a peptide encompassing the Cys-loop sequence of the T. californica subunit, that Asn141 can be
glycosylated before and after oxidation of Cys128 and Cys142 and that
glycosylation of Asn141 actually favors disulfide bond formation. An
additional concern is that X. laevis oocytes apparently are
not capable of complex glycosylation. When T. californica
nAChRs are expressed in X. laevis oocytes, only
oligosaccharides of the high mannose type are incorporated (Buller and
White, 1990). However, although the other glycosylation sites on the
natively expressed T. californica subunit are of the
complex type, glycosylation at the position analogous to Asn141 is of
the high mannose type (Strecker et al., 1994). Whether this
site on 3 receives a high mannose oligosaccharide when this subunit
is expressed by a mammalian neuron is not known, but long term block by
NBT, similar to what we have observed for rat 32 expressed in
oocytes, has been demonstrated with rat sympathetic neurons in culture
(Sah et al., 1987).
The question of proper glycosylation of nAChRs expressed in oocytes
raises the larger issue of whether the pharmacological properties of
nAChRs expressed in oocytes are an accurate reflection of the
properties these receptors display in vivo. We addressed this issue for both muscle and neuronal nAChRs. The agonist
pharmacology of mouse muscle 11 expressed in oocytes
and assayed electrophysiologically (Luetje and Patrick, 1991), was
found to be quite similar to that of the same receptor natively
expressed by BC3H-1 cells (Sine and Steinbach, 1986, 1987). We also
measured the affinity of rat 42 expressed in oocytes for a series
of agonists and antagonists in a radioligand binding using
[3H]cytisine (Parker and Luetje, 1996).
Comparison with the properties of the high affinity cytisine binding
site in rat brain (Pabreza et al., 1991), thought to be
42 (Flores et al., 1992), shows that neuronal nAChRs
expressed in X. laevis oocytes display the same
pharmacological features that these receptors display in a native
context.
Recently, models were proposed for the extracellular domains of
nicotinic receptors (Tsigelny et al., 1997) and glycine
receptors (Gready et al., 1997). Given the homology between
nicotinic and glycine receptor subunits, it seems likely that nAChR
subunit extracellular domains would be structurally similar to the
extracellular domains of glycine receptor subunits. However, these two
models are completely different from each other. The nAChR
extracellular domain model is based on homology with the copper binding
proteins plastocyanin and pseudoazurin, whereas the glycine receptor
extracellular domain model is based on homology with the SH2 and SH3
domains of the Escherichia coli biotin repressor. The
determinants of NBT sensitivity that we have identified fit well with
both models. This is not surprising because both models conform to data
from earlier mutagenesis, antibody mapping, and affinity labeling
studies. Determination of whether either of these models is useful to
the study of ligand-gated channels in general and neuronal nAChRs in
particular will require further mutation analysis.
The exact role of each residue we identified currently is unclear. Each
residue may be interacting directly with NBT. It also is possible that
a residue acts indirectly to change the properties of the binding site
without a direct interaction with NBT. Resolution of this issue will
require manipulation of the toxin structure. The recent use of a
synthetic gene to express recombinant NBT in E.
coli and Pichiia pastoris has allowed structure
function analysis to begin on the NBT molecule (Fiordalisi et
al., 1994, 1991, 1996). A promising approach to identifying
interacting residues on the toxin and the receptor will be the
generation and analysis of compensatory mutations on toxin and
receptor.
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Footnotes |
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Received November 19, 1997; Accepted February 24, 1998
1 Current affiliation: Eli Lilly and Co., Lilly Corporate Center, Indianapolis, IN 46285.
This work was supported by grants to C.W.L. from the National Institute on Drug Abuse (DA08102), the American Heart Association, Florida Affiliate, and the Pharmaceutical Research and Manufacturers of America Foundation. C.W.L. was an Initial Investigator of the American Heart Association Florida Affiliate. S.C.H. was supported in part by T32-HL07188.
Send reprint requests to: Dr. Charles W. Luetje, Department of Molecular and Cellular Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189, Miami, FL 33101. E-mail: cluetje{at}chroma.med.miami.edu
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Abbreviations |
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ACh, acetylcholine;
DHE, dihydro--erythroidine;
nAChR, nicotinic acetylcholine receptor;
NBT, neuronal bungarotoxin;
PCR, polymerase chain reaction;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
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References |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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4.
Neuron
3:
487-496[Medline].9: An acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.
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) and
). Current in response to ACh after
30-min incubation with various concentrations of NBT is presented as a
percentage of the preincubation ACh response (mean ± standard
deviation of three to seven separate oocytes). Some error bars are
obscured by symbols.
), various concentrations of NBT
(
), followed by a 2.5-min wash period, is
presented as a percentage of the preincubation ACh response (mean ± standard deviation of three separate oocytes). Some error bars are
obscured by symbols.
). Current in response to ACh after 30-min incubation
with various concentrations of NBT is presented as a percentage of the
preincubation ACh response (mean ± standard deviation of three to
seven separate oocytes). Some error bars are obscured by symbols. B,
NBT sensitivity of 
, Residues identified in the current study as determinants of NBT
sensitivity.