Identification of critical residues in loop E in the 5-HT3ASR binding site
© Venkataraman et al; licensee BioMed Central Ltd. 2002
Received: 30 January 2002
Accepted: 13 June 2002
Published: 13 June 2002
The serotonin type 3 receptor (5-HT3R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.
Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.
Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, m CPBG, d-tc and lerisetron. 5-HT and m CPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.
Sequence homology between the serotonin type 3 receptor (5-HT3R), the nicotinic acetylcholine receptor (nAChR), the GABAA receptor and the glycine receptor suggests a large amount of structural similarity within this superfamily of ligand gated ion channels[1–3]. This presumed structural homology can be used to guide site directed mutagenesis studies of particular receptor subtypes. On a gross level, all members of this superfamily assemble as pentameric receptors . In some cases, receptors can be assembled from a single subunit (5-HT3AR and nicotinic α7 receptors) [1, 5–8]. In other cases, at least two different subunits are required [2, 4, 9]. The ligand binding site is thought to be formed at the junction between two subunits [2, 4].
Based on data obtained from affinity labeling and site directed mutagenesis studies, several regions of the nicotinic acetylcholine receptor have been implicated in ligand binding . Six essential loops appear to contribute to the binding site with the position of each loop differing slightly depending on whether the receptor is heteromeric or homomeric. For homomeric receptors such as the α7 nAChR, the loops are identified as A, B, C, D, E and F [4, 11]. The amino acids that interact with ligands presumably extend into the binding site from these loops. Subunits are composed of 2 faces (+ and -) with the + face of one subunit forming one side of the binding site and the – face of another subunit forming the complimentary side. Some binding loops (A, B and C) are present on the – face while the remaining loops (D and E) are present on the + face. . The individual amino acids that form binding interactions with the functional groups present on a ligand are likely to differ for each receptor subtype, reflecting the specificity of a particular binding site, however, the overall structure of the binding domain may be similar even for binding sites with different ligand specificities. Identification of ligand specificity requires identification of both the location or structure of individual binding site loops and the amino acids present in a particular receptor subtype.
[3H]Granisetron binding to wildtype and mutant 5-HT3ASRs.
Kd (nM) [3H]granisetron
Bmax (pmol/mg protein)
0.98 ± 0.12
3.2 ± .13
1 ± 0.12
2.7 ± 0.19
0.44 ± .08
1.5 ± 0.14
2.5 ± 0.35
0.32 ± .04
1.3 ± 0.22
4.5 ± 0.5
0.97 ± .14
1.6 ± 0.27
1.9 ± 0.28
1.1 ± .04
0.97 ± 0.13
1.3 ± 0.13
0.81 ± .07
1.38 ± 0.17
2.4 ± 0.34
1.0 ± .16
1.26 ± 0 .21
5.2 ± 1.2
5.5 ± 1.2
0.93 ± 0.19
6.3 ± 0.45
0.56 ± .09
1.19 ± 0 .1
6.9 ± 0.48
4.9 ± 1.1
1.66 ± 0.16
7.8 ± 1.1
0.59 ± .10
1.4 ± 0.24
7.4 ± 0.61
0.30 ± .08
1.51 ± 0.17
Ki values for Inhibition of [3 H]granisetron binding to wildtype and mutant 5-HT3ASRs.
Ki (Inhibition of [3H]granisetron binding) nM
d -tc (nM)
74 ± 17
3.0 ± 0.55
6 ± 2.1
0.8 ± 0.19
97 ± 20
22 ± 6.1
300 ± 120
2.1 ± 0.33
82 ± 9.2
4.1 ± 0.50
4.7 ± 0.87
0.20 ± 0.02
(8 ± 2.0) × 10 3
470 ± 90
39 ± 19
130 ± 28
22 ± 2.2
2.0 ± 0.44
22 ± 2.6
1.5 ± 0.41
29 ± 6.5
0.60 ± 0.10
19 ± 2.6
0.42 ± 0.04
61 ± 14
15 ± 4.9
29 ± 4.9
2.3 ± 0.31
73 ± 11
8.2 ± 1.5
14 ± 1.7
2.8 ± 0.53
170 ± 29
27 ± 3.9
9.3 ± 1.3
1.1 ± 0.21
140 ± 20
1.8 ± 0.18
9.3 ± 1.4
3.7 ± 0.62
(1.8 ± 0.4) × 10 3
73 ± 26
60 ± 19
150 ± 36
270 ± 34
7.7 ± 2.0
17 ± 3.0
3.0 ± 0.66
The Ki value for d-tc inhibition of [3H]granisetron binding was altered only slightly by the Y142A or Y152A mutations (6.5 fold and 10 fold changes respectively). The Y140A mutation, however, produced a 50 fold increase in the Ki compared to wildtype receptors (Figure 4C).
The Ki value for lerisetron inhibition of [3H]granisetron binding was increased 160 fold by the Y142A mutation and 190 fold by the Y152A mutation. Only a 4.6 fold change in Ki resulted from the Y140A mutation. Lerisetron was the only compound for which a large increase in the Ki was observed on the Y152A mutation (Figure 4D). This mutation produced smaller changes in Ki for 5-HT and m CPBG (24 fold for both) and only a 10 fold change for d-tc.
The putative E-loop region of the LGIC family of receptors is homologous to residues Y140 through K153 in the 5-HT3R [4, 12–18]. Structure-function studies of this region have been conducted in several other members of this family of receptors including GABAA and nAChR subtypes. In each case, residues have been identified that alter either the binding of selective ligands or receptor function [4, 11–18]. In order to determine if this loop also contains residues critical to the structure or function of the 5-HT3R, we have constructed alanine mutations of amino acids throughout the homologous region of the mouse 5-HT3ASR and investigated the affects on binding of 5 different structural classes of 5-HT3R ligands. We have identified three tyrosine residues that appear to play a role in binding of selective ligands to this receptor. In addition, our data support the existence of a loop structure in this region as has been hypothesized for the nAChR and identified in a homologous AChBP [12, 23, 24].
Representative members of 5 major structural classes of 5-HT3R ligands were tested on all mutants (Figure 1). These ligands include the antagonists [3H]granisetron, d-tc and lerisetron, and the agonists 5-HT and m CPBG. Most of the mutations tested produced only minor changes in binding affinity for these ligands. Large changes in binding are only apparent on Y140A, Y142A and Y152A receptors for select ligands. The resulting effects of individual mutations are specific to particular structural classes of ligands. The Y140A mutation altered the Ki for d-tc inhibition, but did not alter the Ki obtained for any other compound tested, while Y142A altered the Ki for m CPBG, 5-HT and lerisetron but had little if any effect on inhibition by d-tc. These data indicate the highly specific nature of the effects introduced by the alanine mutations and appear to reflect specific changes in ligand/receptor interaction.
Binding of [3H]granisetron is altered only slightly by the alanine mutations introduced in this study (<10 fold). This result indicates the lack of involvement of amino acids in this binding loop in the binding of [3H]granisetron. It is apparent, however, that [3H]granisetron does occupy the same binding cleft as other 5-HT3R ligands as evidenced by the ability of 5-HT, m CPBG, d-tc and lerisetron to displace it from the binding site. The lack of any large change in binding of granisetron supports our contention that there is little global structural perturbation of the binding site resulting from the introduction of each individual alanine mutation.
The Ki for inhibition of [3H]granisetron binding by 5-HT was increased 110 fold by the Y142A mutation. A similar increase was also observed for m CPBG (160 fold). In contrast, the Y140A and Y152A mutations produced relatively small changes in the Ki. Since both compounds are 5-HT3R agonists, they are likely to share the same binding interactions. One of these interactions appears to be with Y142. Other studies have also identified binding site interactions for agonists. R91, E106, F107, W183 and several residues adjacent to the M1 region have all been demonstrated to alter the action of 5-HT and/or m CPBG [20, 21, 25, 26]. Within the crystal structure of the AChBP, homologous residues are located in the apparent binding site . The effects of agonist on the 5-HT3R are mediated by their interaction with these binding site residues, resulting in stable receptor conformations, including the channel open state. Identification of interacting amino acids and their location in the tertiary structure of the receptor may provide clues to the mechanism of channel opening. For example, amino acids homologous to Y142 and W183 (R104 and W143 respectively) are in close proximity in the AChBP, although on complementary faces of the receptor subunits . Spier et. al. have suggested that W183 may be involved in a cation-π interaction with the amino group of 5-HT and m CPBG . If W183 and Y142 are located near each other but on opposite faces of the binding site in the 5-HT3R, then they could potentially act in concert to help stabilize a conformation of the receptor leading to channel opening.
d-Tubocurarine inhibition was uniquely altered by the Y140A mutation. An increase in Ki of 50 fold was observed for d-tc on this mutant. Little if any change resulted for any other mutation. Thus, while d-tc may form an interaction with the receptor at this binding loop, it appears to interact with Y140 rather than Y142 or Y152. This difference in binding site interactions of the antagonist d-tc and the agonists 5-HT and m CPBG could be the result of a slightly different positioning of d-tc in the binding site. The antagonists d-tc and [3H]granisetron also interact differently with this region since [3H]granisetron binding was not altered by mutations at any of the amino acids tested. While the binding of d-tc appears to involve some interaction with this binding loop, the binding of [3H]granisetron does not. Previous studies have indicated a point of overlap between [3H]granisetron and d-tc at W89 of the 5-HT3ASR. . These studies indicate that the binding regions for these antagonists are partially overlapping at W89 but not at Y140. In addition to Y140 and W89, d-tc has also been observed to interact with D206 and several other residues in loop C . The d-tc binding site appears to involve interaction with at least three different binding loops (A, E and C) although additional interacting amino acids may be found. Data obtained for d-tc is particularly valuable due to the rigid nature of this molecule. Determination of interacting functional groups and their relationship to individual amino acids could enable d-tc to be used as a molecular ruler to determine relative positions of these amino acids.
Lerisetron inhibition of [3H]granisetron binding to mutant receptors was also investigated. Lerisetron is a potent 5-HT3R antagonist first synthesized by Orales et. al.. As was observed for 5-HT3R agonists, lerisetron binding was altered by the Y142A mutation. This mutation produced an increase in the Ki of 160 fold compared to wildtype receptors. This increase in Ki indicates an important interaction of lerisetron with Y142 and a similarity between the binding location of lerisetron, 5-HT and m CPBG. Lerisetron is the only antagonist tested that shares a binding site interaction in this region with agonists. In contrast to 5-HT and m CPBG, however, lerisetron also interacts with Y152 as indicated by the 190 fold increase in Ki on Y152A mutant receptors. Since neither d-tc nor [3H]granisetron interacts with Y142 or Y152, there appears to be a difference between the interactions formed by these antagonists compared to lerisetron. The binding sites of the three antagonists tested differ with respect to this binding loop. Since the role of a competitive antagonist is simply to block the binding of agonists and prevent channel opening, the specific amino acids that interact with the ligand can vary for different antagonists. This is less likely to be the case with agonists since they must produce a conformational change in the protein to exert their effects.
Only two mutant receptors failed to bind [3H]granisetron; G147A and V149A. G147 is the conserved glycine in this putative binding loop. Chirara et. al. have suggested that the highly conserved nature of the glycine in this region may indicate the existence of a loop structure consisting of either γ or a loose three residue-turn in the nAChR . Either of these turns would bring the two putative β-strands together such that γL109, γY111 and γS115 and γY117 are all on the same side of an antiparallel β-sheet. These residues have been identified by affinity labeling, site-directed mutagenesis or cysteine substitution to lie on the same surface. A classic 2-residue β-turn would place these residues on opposite surfaces . Substitution of the conserved glycine by alanine may disrupt the structure of this region and prevent assembly or expression of the receptor. The recent determination of the crystal structure of an AChBP supports this hypothesis. The AChBP displays a large amount of homology to the amino terminal of LGIC receptors and thus may be similar in structure [20, 24]. The crystal structure of this protein reveals a loose 3 residue turn incorporating the conserved glycine residue . Homologous residues in other LGIC subunits have also been identified and are shown in Figure 1. The residues identified in this study as altering binding affinity of 5-HT3R ligands would also be present on the same surface if this structure is present in the 5-HT3R. While Y140 lies somewhat outside the region identified by Chiara in the nAChR (homologous to γN107), γL109 and γL119 are homologous to Y142 and Y152 of the 5-HT3ASR. The ability of lerisetron to interact with both Y142 and Y152 also supports the hypothesis that these two amino acids are present in a loop structure since the eight intervening residues would position Y142 and Y152 too far apart to permit them both to interact with a single ligand even if they were interacting with functional groups on opposite ends of the molecule. A loop structure would bring them into closer proximity and permit interaction with the small molecule lerisetron.
All three tyrosine mutations were investigated using a whole cell patch clamp assay to determine if functional changes could be observed. Whole cell responses could not be obtained for Y140A or Y142A, although specific binding of [3H]granisetron was observed. These data suggest that, while the receptors do assemble and are capable of binding [3H]granisetron, they are either not transported to the cell surface or are non-responsive to 5-HT at concentrations of 1 mM or less.
Y152A does produce functional channels however they display distinctly altered response kinetics when compared to wildtype receptors. Y152A responses do not show the rapid rise times observed in wt receptors. The extremely slow rise times observed for Y152A receptors may indicate a change in rate constants preceding channel opening. These changes in the rate constants for either agonist binding or channel opening also produce a 140 fold decrease in the observed EC50 for 5-HT activation. The slow rise is followed by a non-desensitizing phase of the response that is dramatically different from the fast desensitization observed for wt 5-HT3ASRs. Lack of desensitization could result from either a stabilization of the open state of the channel or a destabilization of the desensitized state. Mutations of homologous or nearby residues in both the nAChR and GABAA receptors have also been demonstrated to alter the agonist response. Mutation of the homologous residue in the GABAA receptor γ-subunit (T142) to serine altered the efficacy of the agonist Flumazenil, converting it to a partial agonist . In the nAChR, mutation of mouse and rat εP121 to leucine altered both the binding of acetylcholine and the stability of the open state of the channel. εP121 is homologous to P154 in the 5-HT3R and is only two residues away from Y152. The authors of this study concluded that this portion of the acetylcholine binding site was closely linked to the channel opening region of the receptor . It is reasonable to conclude that the homologous region in the 5-HT3R may perform a similar function. The link between an agonist binding domain and a conformational change leading to channel opening is not unexpected since the two must obviously be linked. If binding to this region of the receptor is shown to be a critical step between the binding of agonists and the opening of the channel, further investigation of the amino acids in this loop may provide valuable clues to molecular basis of this process.
Our data indicate an important role for this putative binding site loop in the interaction of the 5-HT3R with different ligands and illustrate the difference in binding of different structural classes of ligands. Each structural class shows different patterns of interaction with amino acids in this region of the receptor. [3H]granisetron does not appear to interact with any of the amino acids tested while d-tc interacts with only Y140, m CPBG and 5-HT with Y142 and lerisetron with both Y142 and Y152. Similar selective effects have been observed on other residues including W183, W89, F107 and E106 among others[20, 21, 25, 26]. Our data in conjunction with those of other laboratories indicates the differences in orientation of different ligands within the same binding cleft. These differences in orientation result in different amino acid/functional group interactions. As the structural detail of these interactions emerges, these differences could potentially be exploited to produce more potent and specific ligands. For example construction of a "hybrid" ligand that combines the interactions of [3H]granisetron with those of d-tc, 5-HT or m CPBG could produce an antagonist with increased affinity due to the additional binding energy of these interactions and a greater specificity since it would utilize more structural features of the binding site.
The requirement of a glycine at position 147 and the ability of lerisetron to interact with both Y142 and Y152 also support the hypothesis that the secondary structure in this region of the receptor is formed by a loop structure. If the loose 3 residue turn proposed by Chiara et. al. and shown for the AChBP is present in the 5-HT3R, then Y140, Y142 and Y152 would be present on the same side of the sheet and all three would be capable of extending into the binding site. A similar observation has been made for the nAChR where γS111, γY117, γL119, δR113 and δT119 of the mouse nAChR receptor and γL109 and γY111 of the torpedo nAChR are all thought to be present in the binding site [12–18].
Materials and Methods
[3H]granisetron was purchased from New England Nuclear, 5-HT from Spectrum, and m CPBG and d-tc from Research Biochemical International. Lerisetron was provided by Dr. Karen Kirschbaum at The University of Louisiana at Monroe, Monroe, LA. All other reagents were obtained from commercial sources.
Site directed mutagenesis
Wild type 5-HT3AS mouse receptor cDNA was obtained from Dr. Michael White . Mutant receptors were constructed using either the Quick Change Mutagenesis kit (Stratagene) or the Altered Sites Mutagenesis kit (Promega). All mutations were confirmed by commercial DNA sequencing.
Cell culture and transfection
tsA201 cells, a derivative of HEK293 cells, were seeded at a density of 5 × 106 cells/100 mm dish. Cells were grown in DMEM medium containing 10% FBS, 100 units/ml penicillin/streptomycin for nine hours in 5% CO2 and transfected with 10 μg mouse 5-HT3ASR cDNA per 100 mm dish using the calcium phosphate technique (New Life Technologies, NY). Media was changed 12–14 hrs after transfection. The cells were allowed to grow for another 24 hours and then harvested.
For whole cell patch clamp experiments, tsA201 cells were seeded to a density of 0.25 × 106 cells/60 mm dish. Cells were grown in DMEM culture medium containing 10% FBS and 100 units/ml penicillin/streptomycin for 12 hours prior to transfection. Transfections were performed using Superfect Transfection Reagent (Qiagen, CA). Ten μg of cDNA were mixed with DMEM medium containing no serum or antibiotics in a volume of 150 μl. Twenty μl of Superfect reagent were then added and the mixture incubated at room temperature for 15 min. The reaction was terminated by adding 1 ml of DMEM medium containing 10% FBS and 100 units/ml penicillin/streptomycin and the entire mixture added to cells in the 60 mm dish. Cells were exposed to Superfect Reagent for 3 hours. At that time, the reagent was replaced with DMEM medium containing 10% FBS and 100 units/ml penicillin/streptomycin and incubated for an additional 24 hours prior to use.
Transfected cells were scraped from the dishes, washed twice with Dulbecco's PBS (New Life Technologies, NY), then resuspended in 1.0 ml ice cold PBS/100 mm dish. Cells were either used fresh or frozen at this step until needed. Immediately prior to use, cells were homogenized on ice in PBS using a glass tissue homogenizer then centrifuged at 35 000 × g for 30 minutes in a Beckman JA20 rotor (4°C). Membranes were washed once more with PBS at 4°C then resuspended in 1 ml PBS/100 mm dish. Protein content was determined using a Lowry assay (Sigma. Diagnostics, St. Louis, MO). Membranes were initially prepared and Bmax and Kd values determined in the presence and absence of a cocktail of protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Diagnostics, Mannheim Germany). No change in Bmax was observed as a result of omitting the protease inhibitor (Bmax (+ protease inhibitor)= 3.1 ± 0.11 pmoles/mg protein vs Bmax (- protease inhibitor) = 3.2 ± 0.13 pmoles/mg protein.) hence all assays were performed in the absence of the cocktail. Binding assays were performed in PBS.
For Kd determinations, 100 μl of homogenate was incubated at 37°C for 1 hour with varying concentrations of [3H]granisetron (NEN, MA). Specific binding of [3H]granisetron was determined as the bound [3H]granisetron not displaced by a saturating concentration of a competing ligand. Kd values were determined by fitting the binding data to the following equation using Graphpad PRISM (San Diego CA): B = Bmax [L]n / ([L]n + Kn), where B is bound ligand, Bmax is the maximum binding at equilibrium L is the free ligand concentration and n is the Hill coefficient.
Ki determinations, 100 μl of homogenate was incubated at 37°C for 2 hours with varying concentrations of inhibitor and [3H]granisetron (NEN, MA). Binding was terminated by rapid filtration onto a GF/B filters. The IC50 values were calculated by fitting the data to the following equation using Graphpad PRISM (San Diego CA): θ = 1/ (1+(L/IC50)), where θ is the fractional amount of [3H]granisetron bound in the presence of inhibitor at concentration L as compared to the amount of [3H]granisetron bound in the absence of inhibitor. IC50 is the concentration at which θ = 0.5. The Ki is calculated from the IC50 value using the Cheng-Prusoff equation.
Transfected cells were transferred to a recording chamber containing extracellular solution (140 mM NaCl, 1.7 mM MgCl2, 5 mM KCl, 1.8 mM CaCl2, 25 mM HEPES pH 7.4). Patch electrodes of resistance 2.5–3.0 MΩ were filled with filtered intracellular solution containing 145 mM KCl, 2 mM MgCl2, 1 mM EGTA, 25 mM HEPES (pH 7.4). Cells were clamped in whole cell configuration at a holding potential of -60 mV. A continuous extracellular solution flow (0.8 ml/min) was maintained throughout the recording procedure. 5-HT was dissolved in extracellular solution and delivered to cells using a rapid perfusion system (Warner Instruments, Hamden, CT) at a rate matching the extracellular solution flow rate. The drug perfusions lasted for a period varying from 4 to 8 seconds. Currents elicited by agonist application were measured using an Axopatch 200 B amplifier (Foster City, CA). The data were plotted and analyzed by non-linear curve fitting (Graphpad PRISM, San Diego CA) according to the following equation: I = 1/(1 +(EC50/ [C])n), where is the normalized current at 5-HT concentration [C], EC50 is the concentration of 5-HT needed to obtain half maximal activation and n is the apparent Hill coefficient.
This work was supported by The University of Louisiana at Monroe, College of Pharmacy and grants from the Southeast Affiliate of the American Heart Association. We would also like to gratefully acknowledge Dr. Karen Kirschbaum at The University of Louisiana at Monroe, Monroe, LA for providing us with the lerisetron used in these studies.
- Maricq AV, Peterson AS, Brake AJ, Myers RM, Julius D: Primary structure and functional expression of the 5-HT3R, serotonin gated ion channel. Science. 1991, 397: 359-363.Google Scholar
- Karlin A, Akabas MH: Towards a structural basis for the function of nicotinic acetylcholine recetors and their cousins. Neuron. 1995, 15: 1231-1244.PubMedView ArticleGoogle Scholar
- Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Rmachandran J, Reale V, Glencorse TA, Seeburg PH, Barnhard EA: Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature. 1987, 328 (6127): 221-227. 10.1038/328221a0.PubMedView ArticleGoogle Scholar
- Arias HR: Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors. Neurochem Intl. 2000, 36: 595-645. 10.1016/S0197-0186(99)00154-0.View ArticleGoogle Scholar
- Hope AG, Downie DL, Sutherland L, Lambert JJ, Peters JA, Burchell B: Cloning and functional expression of an apparent splice variant of the murine 5-HT3 receptor A subunit. E. J. Pharm. 1993, 245: 187-192. 10.1016/0922-4106(93)90128-V.View ArticleGoogle Scholar
- Belelli D, Balcarek JM, Hope AG, Peters JA, Lambert JJ, Blackburn TP: Cloning and functional expression of a human 5-hydroxytryptamine type 3AS receptor subunit. Mol. Pharm. 1995, 48: 1954-1062.Google Scholar
- Couturier S, Bertrand D, Matter JM, Hernandez MC, Bertrand S, Millar N, Valera S, Barkas T, Ballivet M: A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron. 1990, 5 (6): 847-56.PubMedView ArticleGoogle Scholar
- Seguela P, Wadiche J, Dineley-Miller K, Dani JA, Patrick JW: Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J Neurosci. 1990, 13 (2): 596-604.Google Scholar
- Dubin AE, Huvar R, D'Andrea MR, Pyati J, Zhu JY, Joy KC, Wilson SJ, Galindo JE, Glass CA, Luo L, Jackson MR, Lovenberg TW, Erlander MG: The pharmacological and functional characteristics of the serotonin 5-HT3A receptor are specifically modified by a 5-HT3B receptor subunit. J. Biol. Chem. 1999, 274 (43): 30799-30810. 10.1074/jbc.274.43.30799.PubMedView ArticleGoogle Scholar
- A Devillers-Thiery, Galzi JL, Eisele JL, Bertrand S, Bertrand D, JP Changeux: Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand gated ion channels. J. Membrane Biol. 1993, 136: 97-112.View ArticleGoogle Scholar
- Corringer P, Le Nove're N, Changeux J: Nicotinic receptors at the amino acid level. Annu. Rev. Pharmacol. Toxicol. 2000, 40: 431-58. 10.1146/annurev.pharmtox.40.1.431.PubMedView ArticleGoogle Scholar
- Chiara DC, Xie Y, Cohen JB: Structure of the agonist-binding sites of the torpedo nicotinic acetylcholine receptor: affinity-labelling and mutational analysis identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants. Biochemistry. 1999, 38: 6689-6698. 10.1021/bi9901735.PubMedView ArticleGoogle Scholar
- Sine SM: Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity. Proc.Natl.Acad.Sci. 1993, 90: 9436-9440.PubMed CentralPubMedView ArticleGoogle Scholar
- Sine SM, Kreienkamp HJ, Bren N, maeda R, Taylor P: Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity. Neuron. 1995, 15: 205-211.PubMedView ArticleGoogle Scholar
- Sine SM: Identification of equivalent residues in the, γ, δ δ and ε subunits of the nicotinic receptor that contribute to α-bungarotoxin binding. J.Biol.Chem. 1997, 272 (38): 23521-23527. 10.1074/jbc.272.38.23521.PubMedView ArticleGoogle Scholar
- Gu Y, Camacho P, Gardner P, Hall ZW: Identification of two amino acid residues in the ε subunit that promote mammalian muscle acetylcholine receptor assembly in COS cells. Neuron. 1991, 6: 879-887.PubMedView ArticleGoogle Scholar
- Kreienkamp HJ, Maeda RK, Sine SM, Taylor P: Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor. Neuron. 1995, 14 (3): 635-644.PubMedView ArticleGoogle Scholar
- Westh-Hansen SE, Rasmussen PB, S Hastrup, J Nabekura, K Noguchi, N Akaike, Witt MR, Nielsen M: Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit. Eur. J. Pharmacol. 1997, 329 (2-3): 253-257. 10.1016/S0014-2999(97)10133-9.PubMedView ArticleGoogle Scholar
- Green T, Stauffer KA, Lummis SCR: Expression of recombinant homo-oligomeric 5-hydroxytryptamine3 receptors provides new insights into their maturation and structure. J. Biol. Chem. 1995, 270 (11): 6056-6061. 10.1074/jbc.270.11.6056.PubMedView ArticleGoogle Scholar
- Yan D, Schulte MK, Bloom KE, White MM: Structural features of the ligand-binding domain of the serotonin 5HT3R. J. Biol. Chem. 1999, 274 (9): 5537-5541. 10.1074/jbc.274.9.5537.PubMedView ArticleGoogle Scholar
- Boess FG, Steward LJ, Steele JA, Liu D, Reid J, Glencorse TA, Martin IL: Analysis of the ligand binding site of the 5-HT3R using site directed mutagenesis: Importance of glutamate 106. Neuropharm. 1997, 36 (4/5): 637-647. 10.1016/S0028-3908(97)00044-0.View ArticleGoogle Scholar
- Lummis SC, Sepulveda MI, Kilpatrick GJ, Baker J: Characterization of [3H]meta-chlorophenylbiguanide binding to 5-HT3 receptors in N1E-115 neuroblastoma cells. Eur J Pharmacol. 1993, 243 (1): 7-11. 10.1016/0014-2999(93)90160-J.PubMedView ArticleGoogle Scholar
- Smit AB, Syed NI, Schaap D, van Minnen J, Klumperman J, Kits KS, Lodder H, van der Schors RC, van Elk R, Sorgedrager B, Brejc K, Sixma TK, Geraerts WPM: A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature. 2001, 411: 261-268. 10.1038/35077000.PubMedView ArticleGoogle Scholar
- Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van der Oost J, Smit AB, Sixma TK: Crystal structure of an Ach-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature. 2001, 411: 269-276. 10.1038/35077011.PubMedView ArticleGoogle Scholar
- Steward LJ, Boess FG, Steele JA, Liu D, Wong N, Martin IL: Importance of phenylalanine 107 in agonist recognition by the 5-hydroxytryptamine3A receptor. Mol. Pharm. 2000, 57: 1249-1255.Google Scholar
- Spier AD, Lummis SCR: The role of tryptophan residues in the 5-hydroxytryptamine3 receptor ligand binding domain. J. Biol Chem. 2000, 275 (8): 5620-5625. 10.1074/jbc.275.8.5620.PubMedView ArticleGoogle Scholar
- Hope AG, Belelli D, Mair ID, Lambert JJ, Peters JA: Molecular determinanats of (+)tubocurarine binding at recombinant 5-hydroxytryptamine3A receptor subunits. Mol. Pharm. 1999, 55: 1037-1043.Google Scholar
- Orjales A, Mosquera R, Labeaga L, Rodes R: New 2-Piperazinylbenzimidazole derivatives as 5HT3 antagonists. Synthesis and pharmacological Evaluation. J. Med. Chem. 1997, 40: 586-593. 10.1021/jm960442e.PubMedView ArticleGoogle Scholar
- Mihic JS, Whiting PJ, Klein RL, Wafford KA, Harris RA: A single amino acid of the human γ-aminobutyric acid type A receptor γ2 subunit determines benzodiazepine efficacy. J. Biol. Chem. 1994, 269 (52): 32768-32773.PubMedGoogle Scholar
- K Ohno, Wang H, Milone M, Bren N, Brengmean JM, Nakano S, Quiram P, Pruitt JN, Nine SM, Engel AG: Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor ε subunit. Neuron. 1996, 17: 157-170.View ArticleGoogle Scholar
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