From the Departments of Pharmacology and Physiology, MCP Hahnemann University, Philadelphia, Pennsylvania 19129
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ABSTRACT |
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The nicotinic acetylcholine receptor (AChR) and
the serotonin type 3 receptor (5HT3R) are members of
the ligand-gated ion channel gene family. Both receptors are inhibited
by nanomolar concentrations of d-tubocurarine (curare) in a
competitive fashion. Chemical labeling studies on the AChR have
identified tryptophan residues on the The serotonin type 3 receptor
(5HT3R)1 is a
member of a superfamily of ligand-gated ion channels, which includes
the muscle and neuronal nicotinic acetylcholine receptor (AChR), the
glycine receptor, and the A large number of studies have been conducted to elucidate the
structure of the ligand-binding site for AChRs (6, 7); however, very
little is known about the interactions between ligands and the
5HT3R. A common competitive antagonist of the AChR and 5HT3R is d-tubocurarine (curare), which inhibits
both receptors at nanomolar concentrations (8, 9). If both receptors
are similar in structure, it seems reasonable to assume that similar regions of the receptors may be involved in curare binding. Affinity labeling studies using the Torpedo electroplax AChR have
identified two tryptophan residues, one on the Comparison of the deduced amino acid sequences of this region of a
number of members of the gene superfamily shows that tryptophan is
found in this position in the AChR Isolation of 5HT3R cDNA and Mutagenesis--
A
pair of oligonucleotides (GCCTACTACTGTCTCCATTGA and
CTCCCACTCGCCCTGATTTAT) derived from the published sequence of the
original 5HT3R cDNA (4) was used to amplify a 460-base
pair fragment from RNA isolated from the murine neuroblastoma line
NIE-115 using reverse transcription-polymerase chain reaction (RNA
polymerase chain reaction kit, P/E/Express). The fragment was then
used to screen a plasmid cDNA library made from N1E-115 mRNA. A
full-length cDNA clone corresponding to the
5HT3RAs form (16) was isolated and subcloned
into vector pCI (Promega, Madison, WI). Site-directed mutagenesis was
performed using the QuickChange system (Stratagene, La Jolla, CA). All
mutants were verified through sequence analysis. Because the amino
terminus of the mature protein is unknown, the amino acid numbering
system used here includes the signal sequence and has the initial
methionine as position 1.
Secondary Structure Analysis--
Protein secondary structure
analysis was carried out using the Chou-Fasman (17) and Garnier-Robson
(18) algorithms contained in a commercially available software package
(Lasergene, DNAStar, Inc., Madison, WI).
Transfection--
Cultures of the tsA201 cell line, a derivative
of the widely used HEK 293 cell line, were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin. Cultures at
30-40% confluence were transfected with 20 µg of 5HT3R
cDNA/100-mm dish using the calcium phosphate technique. After 12-h
exposure to the DNA/calcium phosphate solution, the medium was replaced
with fresh medium, and the cells were allowed to grow for another
24-36 h prior to use. Maximal expression was obtained 36-72 h after transfection.
Ligand Binding Assays--
Transfected cells were scraped from
the dishes, washed once with phosphate-buffered saline and resuspended
and homogenized in 2.5 ml of 154 mM NaCl, 50 mM
Tris-HCl, pH 7.4, per 100-mm dish. The homogenate was then used in
binding assays or frozen until needed. We observed no change in either
ligand affinity or Bmax values after freezing.
Membranes were incubated for 2 h at 37 °C in a total volume of
0.5 ml of 154 mM NaCl, 50 mM Tris-HCl, pH 7.4, containing the appropriate concentrations of the competing ligand and
radioligand ([3H]granisetron; NEN Life Science Products,
85 Ci/mmol). Binding was terminated by rapid vacuum filtration onto
GF/B filters that had been pretreated in 0.2% polyethyleneimine in 50 mM Tris-HCl, pH 7.4, and the filters were washed with cold
50 mM Tris-HCl, pH 7.4. Nonspecific binding was defined as
that binding not displaced by 100 µM
m-chlorophenyl biguanide. Kd values for
[3H]granisetron were determined by fitting the saturation
binding data to Equation 1 using a Levenberg-Marquardt algorithm in a commercially available software package (Igor Pro, WaveMetrics, Oswego,
OR),
Electrophysiology--
Transfected cells were transferred to
35-mm dishes containing 140 mM NaCl, 1.7 mM
MgCl2, 5 mM KCl, 1.8 mM
CaCl2, 25 mM HEPES, pH 7.4, and currents
elicited by bath application of agonists at a holding potential of
Dose-response curves from individual cells were normalized to the
maximum current and were fit to Equation 4 as described previously
(21),
Our initial focus was on Trp-89, which is in the homologous
position to the two tryptophan residues in the Torpedo AChR
that are photolabeled by curare, (
Trp-55) and
(
Trp-57) subunits that interact with curare. Comparison of the
sequences of these two subunits with the 5HT3R shows that a
tryptophan residue is found in the homologous position in the
5HT3R (Trp-89), suggesting that this residue may be
involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the
5HT3R for the antagonists curare and granisetron but has
little effect on the affinity for the agonist serotonin. To further
examine the role of this region of the receptor in ligand-receptor
interactions, alanine-scanning mutagenesis analysis of the region
centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand
binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only
at Trp-89, whereas serotonin affinity is reduced only by substitution
at Arg-91. On the other hand, granisetron affinity is reduced by
substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect
of substitutions on ligand affinity suggests that different ligands may
have different points of interaction within the ligand-binding pocket.
In addition, the every-other-residue periodicity of the effects on
granisetron affinity strongly suggests that this region of the
ligand-binding site of the 5HT3R (and by inference, other
members of the ligand-gated ion channel family) is in a
-strand conformation.
INTRODUCTION
Top
Abstract
Introduction
References
-aminobutyric acid type A receptor (1-3). Like the other members of the gene superfamily, the 5HT3R
exhibits a large degree of sequence similarity, and thus presumably
structural homology, with the AChR (4). Chimeras containing the
amino-terminal domain of the
7 neuronal AChR and the
carboxyl-terminal domain of the 5HT3R form functional
ligand-gated channels with ligand specificities characteristic of AChR
and permeability properties of 5HT3Rs (5). These
experiments suggest that the AChR and 5HT3R have quite
similar structures and signal transduction mechanisms.
(
Trp-55) and the
other on the
(
Trp-57) subunit, as being involved in curare-AChR
interactions (10). Mutagenesis data are consistent with
Trp-55
contributing to the high affinity curare-binding site (11), which is
known to exist at the
-
subunit interface (12, 13).
(
Trp-55) and
(
Trp-57) subunits and the 5HT3R (position Trp-89) but not most other
receptors (Fig. 1). In support of the
notion that Trp-89 may contribute to the curare-binding site of
5HT3Rs, chemical modification studies have suggested that
one or more tryptophan residues contribute to the binding of a number
of 5HT3R ligands (14). To determine the role of Trp-89 in
ligand-5HT3R interactions, we have carried out an alanine
scan mutational analysis (15) of the region around Trp-89. We have
identified several residues in this region that play a role in
ligand-receptor interactions and have further identified residues that
may make differential contributions to agonist and antagonist binding.
Furthermore, the periodicity of the effects of the mutations on ligand
affinity suggests that this particular region of the ligand-binding
domain is in a
-strand conformation.
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Fig. 1.
Ligand-gated ion channel sequence alignments
of the region surrounding the tryptophans in the acetylcholine receptor
photolabeled by curare. Sequences centered on residues in
positions homologous to Trp-55 in the and Trp-57 in the
subunit
of the AChR for the indicated receptor subunits are shown. Note that
tryptophan is found only in the AChR
and
subunits and the
5HT3R. GlyR, glycine receptor;
GABAAR,
-aminobutyric acid type A receptor.
EXPERIMENTAL PROCEDURES
where B is the amount of
[3H]granisetron specifically bound at concentration
[L], Bmax is the maximal binding at
saturation, and Kd is the dissociation constant.
IC50 values for various inhibitors were determined by
fitting the data to Equation 2,
(Eq. 1)
where
(Eq. 2)
is the fractional amount of
[3H]granisetron bound in the presence of the inhibitor at
concentration [I] compared with that in the absence of inhibitor and
IC50 is the concentration of inhibitor at which
= 0.5. Ki values were calculated from the IC50
values using the Cheng-Prusoff relation (19) (Equation 3),
where [L] is the concentration of
[3H]granisetron used to determine the IC50
value in the experiment and Kd is the dissociation
constant for [3H]granisetron.
(Eq. 3)
70
mV were measured in the whole-cell configuration (20) with an Axopatch
200A amplifier (Axon Instruments, Foster City, CA) under computer
control using custom programs written in AxoBasic. Patch electrodes
contained 145 mM KCl, 2 mM MgCl2, 1 mM EGTA, 25 mM HEPES, pH 7.4, and had
resistances of 3-5 megaohms. Agonist and antagonists were dissolved in
extracellular solution and delivered to the cell by means of a fast
perfusion system (Warner Instruments, Hamden, CT). For experiments
involving inhibition of 5-HT-elicited currents by curare, cells were
perfused with the desired concentration of curare for 2 min before
switching to a solution containing 5-HT and curare.
where
(Eq. 4)
is the normalized current at 5-HT concentration
[A], EC50 is the concentration of 5-HT
required to obtain half-maximal current, and n is the
apparent Hill coefficient.
RESULTS
Trp-55 and
Trp-57 (10).
Initially, Trp-89 was replaced by alanine, which should have little
effect on
-helical or
-sheet structure and thus should produce
little structural perturbation. However, we were unable to elicit whole cell currents with 5-HT concentrations up to 10 mM from
cells transfected with W89A receptors nor were we able to detect
specific binding of the antagonist [3H]granisetron in
membranes prepared form transfected cells (data not shown). Obviously,
substitution of Trp-89 with alanine either interfered with the proper
assembly or the functioning of the receptor. We then replaced
tryptophan with another aromatic amino acid, phenylalanine, which
should represent a less severe substitution. Fig.
2 shows 5-HT-elicited currents recorded
from cells transfected with either wild type (Fig. 2, left)
or W89F (Fig. 2, right). Although the activation properties
of the two receptors are essentially identical (Table
I), the sensitivity to the competitive
antagonist curare was markedly different. Wild type receptors were
inhibited 85% by 10 nM curare, whereas W89F receptors were
inhibited less than 15% under the same conditions.
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Fig. 2.
Amino acid substitution at position Trp-89
markedly reduces curare sensitivity. Cells expressing either wild
type (left) or W89F 5HT3Rs were voltage-clamped,
and currents elicited by bath application of 1 µM 5HT in
the presence or absence of 10 nM curare (dTC)
were recorded. Note that the mutant receptors are relatively
insensitive to curare.
Properties of wild type and W89F 5HT3Rs
This reduction in curare sensitivity is also seen in ligand binding studies. Saturation binding isotherms for the competitive antagonist [3H]granisetron were determined for both wild type and W89F receptors (Fig. 3A). The Bmax value for wild type receptors was on the order of 2-4 pmol/mg of protein, whereas that of the W89F receptors was 1-2 pmol/mg of protein (data not shown). The expression levels of wild type receptors are 5-10 times that reported for N1E-115 neuroblastoma cells (22) or transiently transfected COS-1 (4) cells. The affinity of the W89F receptors for [3H]granisetron was markedly reduced compared with wild type (Kd = 10.9 ± 2.9 nM (W89F) versus 1.3 ± 0.1 nM (wild type)).
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We examined the effects of the W89F mutation on the affinity of other agonists and antagonists through the use of analysis of the reduction in [3H]granisetron binding in the presence of increasing concentrations of agonists and antagonists. Inhibition of [3H]granisetron binding by curare to wild type and W89F receptors showed a similar reduction in ligand affinity in the mutant (Fig. 3B). The Ki for curare inhibition in wild type receptors was 138 ± 22 nM, whereas the value for W89F receptors was 1063 ± 179 nM, a reduction in affinity comparable with that seen in the [3H]granisetron saturation binding isotherms. A similar reduction in affinity was also observed for another 5HT3R antagonist, MDL-72222 (data not shown). In all three cases, the W89F mutation results in a reduction of ligand affinity. However, when agonists were used in the competition studies, a different picture emerges. No significant difference in affinity between mutant and wild type receptors was observed for the full agonist serotonin (Fig. 3C) or the partial agonists phenylbiguanide or m-chlorophenylbiguanide (data not shown). The serotonin data are consistent with the electrophysiological assays in which no difference in the activation parameters was observed between wild type and W89F receptors.
The above findings suggest that Trp-89 interacts specifically with
antagonists but not with agonists. To explore this in more detail,
alanine-scanning mutagenesis (15) of this region of the receptor was
carried out. In this approach, functionally important residues are
identified by sequential replacement of residues within a specific
region with alanine. Alanine is used as the replacement because it
eliminates the side chain beyond the -carbon yet, unlike glycine,
generally does not alter main chain conformation. We carried out the
analysis on nine residues flanking Trp-89 (Thr-85 to Trp-94) using
[3H]granisetron, curare, and serotonin as the ligands to
examine the properties of the binding site. All nine
alanine-substituted receptors were expressed on the cell surface and
formed 5HT3Rs. The receptor density (determined as the
Bmax values for [3H]granisetron
binding) of the mutant receptors ranged from 0.1 pmol/mg (Q92A) to 2.2 pmol/mg (T86A).
The results of the alanine scan in terms of ligand affinities are presented in Fig. 4. Each of the three ligands shows a distinct profile. For example, the only substitution that significantly affects the interaction of curare with the receptor is W89F; all other replacements in this region have little effect on affinity. In the case of the agonist serotonin, the only substitution that alters the affinity of the receptor is R91A. Finally, the affinity of [3H]granisetron is reduced by substitutions at both Trp-89 and Arg-91, as well as by a third, Y93A. These results suggest that whereas this region of the receptor is clearly involved in ligand binding, each of these three structurally different ligands interacts in a slightly different fashion with the ligand-binding domain of the receptor. These differences, in turn, can provide some information on some of the structural features of the receptor.
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DISCUSSION |
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Previous studies have suggested the involvement of tryptophan
residues in ligand binding in both the AChR (10, 11) and the
5HT3R (14). The data presented in this study demonstrate that Trp-89, which is in the same homologous position as the two tryptophan residues in the AChR that interact with curare (Trp-55,
Trp-57), plays an important role in curare-5HT3R
interactions. The fact that this is a region in the 5HT3R
homologous to a domain known to be involved in ligand binding in the
AChR lends support to the notion that similar regions in the two
receptors are involved in ligand binding.
The data presented in this study suggest that although the region around Trp-89 in the 5HT3R is clearly involved in ligand binding, the specific interactions of various compounds with the binding site may be different. In particular, the fact that in the region Thr-85 to Trp-94 substitution at Trp-89 affects curare, but not serotonin, affinity indicates that there are differential interactions between the binding site and the ligands. This is presumably because of differences in ligand structure and/or positioning within the binding site.
In addition to the notion that the "fine structure" of
ligand-receptor interactions may differ for different ligands, our data
also provide some insight into the structure of the binding site
itself. Unlike curare and serotonin, whose binding is sensitive to
mutations at only one residue, the affinity of the antagonist granisetron is affected by mutations at three different residues in
this region (Trp-89, Arg-91, Tyr-93). The fact that the relevant positions are in an every-other-residue pattern suggests very strongly
that this region of the receptor is in a -strand conformation. In
support of this hypothesis, secondary structure predictions using the
Chou-Fasman (17) and Garnier-Robson (18) algorithms predict that this
region of the protein should exist in a
-sheet structure. This
aspect of the study demonstrates the power of the alanine scan
mutagenesis approach, which can pick up structural features that a more
focused single-residue mutagenesis approach might miss.
Tsigelny et al. (23) have described a model for the
extracellular domain of the nicotinic acetylcholine receptor that is based on sequence similarity to copper-binding proteins of known crystal structure. The region homologous to the domain that we have
studied (residues 51-60 in their numbering system) was modeled as an
-helical structure by these authors. However, this region was in one
of the four insertions that had to be introduced into the backbone of
the copper-binding protein scaffold, and their assignment was based on
the fact that in their hands the Chou-Fasman indicates a favorable
environment for
-helical structure. In our secondary structure
analysis, we did notice that the Chou-Fasman algorithm predicted an
-helical structure for several small stretches in this region of the
5HT3R but that the helical prediction was weaker than that
for a
-strand, which extended through the entire region.
Furthermore, an alternate prediction algorithm, the Garnier-Robson method, did not predict any
-helical structure in this region but
did predict a
-strand structure. In addition, our own analysis of
this particular region of the AChR using the Chou-Fasman and Garnier-Robson algorithms indicated that this particular region of the
and
subunits of the AChR should be in the
-strand, not
-helical, configuration. In any case, we feel that our experimental data strongly favor a
-strand structure for this region of the 5HT3R and, on homology grounds, in all other members of the
family, including the AChR.
The data presented here are consistent with the notion that there is a
significant amount of structural and functional homology between the
AChR and 5HT3R (and by inference, the other members of the
ligand-gated ion channel family). Changeux and co-workers (6) have
proposed that the ligand-binding domain of the AChR is formed by
several distinct regions of the and non-
subunits. We envision
that the ligand-binding domain of the 5HT3R is also formed
by several different parts of the extracellular domain that are
homologous to those regions of the AChR known to be involved in ligand
binding. The present study has focused on one of these regions, and the
data indicate that this notion that homologous regions are involved in
ligand binding is indeed correct. The expansion of our studies to the
other regions of 5HT3R that are homologous to the
ligand-binding domains of the AChR will allow us to test the notion
that the functional homology of the members of the ligand-gated ion
channel family extends to a fairly resolved level. Given the success of
our initial foray, as well as our findings concerning the secondary
structure of a portion of the binding site, we envision this as a
somewhat reciprocating process, in which information obtained from one
member of the gene family can be used to guide the studies on another,
and vice versa.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01 NS23885 and T32 NS09618.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.
Present address: Division of Biochemical Pharmacology, School of
Pharmacy, Northeast Louisiana University, 700 University Ave.,
Monroe, LA 71209.
§ To whom correspondence should be addressed: Depts. of Pharmacology and Physiology, MCP Hahnemann University, 3200 Henry Ave., Philadelphia, PA 19129. Tel.: 215-842-4750; Fax: 215-843-4719; E-mail: mwhite{at}mcphu.edu.
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ABBREVIATIONS |
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The abbreviations used are: 5HT3R, serotonin type 3 receptor; AChR, nicotinic acetylcholine receptor.
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REFERENCES |
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