Micromechanical measurement of AChBP binding for label-free drug discovery.

A potential binding assay based on binding-driven micromechanical motion is described. Acetylcholine binding protein (AChBP) was used to modify a microcantilever. The modified microcantilever was found to bend on application of the naturally occurring agonist (acetylcholine) or the antagonist (nicotine and d-tubocurarine). Control experiments show that microcantilevers modified without AChBP do not respond to acetylcholine, nicotine, and d-tubocurarine. K(d) values obtained for acetylcholine, nicotine, and d-tubocurarine are similar to those obtained from radio-ligand binding assays. These results suggest that the microcantilever system has potential for use in label free, drug screening applications.

Mapping spatial relationships between residues in the ligand-binding domain of the 5-HT3 receptor using a molecular ruler.

The serotonin 5-HT(3) receptor (5-HT(3)R) is a member of the Cys-loop ligand-gated ion channel family. We used a combination of site-directed mutagenesis, homology modeling, and ligand-docking simulations to analyze antagonist-receptor interactions. Mutation of E236, which is near loop C of the binding site, to aspartate prevents expression of the receptor on the cell surface, and no specific ligand binding can be detected. On the other hand, mutation to glutamine, asparagine, or alanine produces receptors that are expressed on the cell surface, but decreases receptor affinity for the competitive antagonist d-tubocurarine (dTC) 5-35-fold. The results of a double-mutant cycle analysis employing a panel of dTC analogs to identify specific points of interactions between the dTC analogs and E236 are consistent with E236 making a direct physical interaction with the 12 -OH of dTC. dTC is a rigid molecule of known three-dimensional structure. Together with previous studies linking other regions of dTC to specific residues in the binding site, these data allow us to define the relative spatial arrangement of three different residues in the ligand-binding site: R92 (loop D), N128 (loop A), and E236 (near loop C). Molecular modeling employing these distance constraints followed by molecular-dynamics simulations produced a dTC/receptor complex consistent with the experimental data. The use of the rigid ligands as molecular rulers in conjunction with double-mutant cycle analysis provides a means of mapping the relative positions of various residues in the ligand-binding site of any ligand-receptor complex, and thus is a useful tool for delineating the architecture of the binding site.

Anesthesia in adult cardiac surgery without maintenance of muscle relaxants: a randomized clinical trial.

There may be no need for muscle paralysis during cardiac surgery when adequate anesthesia is provided. We studied intra- and post-operative conditions during cardiac surgery without maintenance muscle relaxant therapy. Eighty adult patients who were candidates for elective coronary artery bypass graft surgery were randomly allocated into two groups. In the noMR or study group (noMR group; n = 40) only an intubation dose of cisatracurium (0.15 mg kg(-1)) was administrated, as opposed to the control group (MR group; n = 40), who had a continuous infusion added to the intubation dose. The anesthesia level was maintained at a Bispectral score of 40-50 using a propofol infusion. A remifentanil infusion was titrated to control patient hemodynamic response. During surgery, any minor (fine body or respiratory muscle movements) or major (coarse body movements or bucking/caught) movements were recorded. Postoperatively, analgesia was provided by remifentanil. The surgical condition was classified into three states: good (no movement), acceptable (minor movements), or poor (major movements). Anesthesia, surgery and postoperative characteristics were compared between the two groups. Statistical analysis was performed in only 78 patients (noMR = 38, MR = 40). The demographic and preoperative characteristics of the two groups were comparable. Intra-operative propofol consumption was the same, but significantly more remifentanil was used in the noMR group (p = 0.001). Post-operative characteristics and complication rates did not differ between the two groups. There were no movements in the MR group patients, while in the noMR group one patient had major movement and three had minor movements. We concluded that omitting maintenance muscle relaxants in adult cardiac surgery or eliminating residual muscle paralysis at the end of the surgery without improving early outcome can increase patient intra-operative movement risk.

Tryptophan 86 of the alpha subunit in the Torpedo nicotinic acetylcholine receptor is important for channel activation by the bisquaternary ligand suberyldicholine.

Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

Mapping residues in the ligand-binding domain of the 5-HT(3) receptor onto d-tubocurarine structure.

The serotonin 5-HT(3) receptor (5-HT(3)R) is a member of the cys-loop ligand-gated ion channel family. We have used the combination of site-directed mutagenesis, homology modeling of the 5-HT(3)R extracellular domain, and ligand docking simulations as a way to map the architecture of the 5-HT(3)R ligand binding domain. Mutation of Phe226 in loop C of the binding site to tyrosine (F226Y) has no effect on the apparent affinity of the competitive antagonist d-tubocurarine (dTC) for the receptor. On the other hand, replacement of Asn128 in loop A of the binding site with alanine (N128A) increases the apparent affinity of dTC by approximately 10-fold. Double-mutant cycle analysis employing a panel of dTC analogs with substitutions at various positions to identify specific points of interactions between the dTC analogs and Asn128 suggests that Asn128 makes a direct interaction with the 2'N of dTC. Molecular modeling of the 5-HT(3)R extracellular domain using the antagonist-bound conformation of the Aplysia californica acetylcholine binding protein as a template followed by ligand docking simulations produces two classes of structures of the 5-HT(3)R/dTC complex; only one of these has the 2'N of dTC positioned at Asn128 and thus is consistent with the data from this study and previously published data. The use of the rigid dTC analogs as "molecular rulers" in conjunction with double-mutant cycle analysis of mutant receptors can allow the spatial mapping of the position of various residues in the ligand-binding site.

Thymosin alpha1 suppresses proliferation and induces apoptosis in human leukemia cell lines.

Thymosin alpha1 (Talpha1), a 28-amino acid peptide, is a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Talpha1 on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562 and K562/ADM) were studied. The proliferation was significantly depressed after 96 h of treatment with Talpha1, and obvious signs of apoptosis, i.e., cell morphology, nuclei condensation and Annexin V binding, were observed thereafter. Moreover, the up-regulation of Fas/Apol (CD95) and decrease in bcl-2 anti-apoptotic gene expression were observed in apoptotic cells. The expression and the function of P-glycoprotein (P-gp) can be slightly inhibited by Talpha1. It is noteworthy that K562 and K562/ADM were more sensitive than HL-60 cells when subjected to Talpha1. Furthermore, HepG-2, the human hepatoma cell line, displayed significant less sensitivity to Talpha1 than all the human leukemia cell lines. D-Tubocurarine (TUB), a nicotinic acetylcholine receptors (nAChRs) antagonist, significantly antagonized the inhibition effects induced by Talpha1, whereas atropine, a muscarinic acetylcholine receptor antagonist, did not exhibit such effects. All the results indicate that Talpha1 was able to significantly suppress proliferation and induce apoptosis in human leukemia cell lines.

Efecto de la atorvastatina en la expresión de Fas (CD95/Apo-1) en tejido cerebral normal e isquémico / Effect of atorvastatin on Fas (CD95/Apo-1) expression in normal or ischemic brain

Introducción: La fisiopatología de la isquemia cerebral involucramúltiples eventos, entre ellos, importantes mecanismos apoptóticos dondela proteína Fas es expresada. Las estatinas poseen efectos neuroprotectoresindependientes de sus acciones hipolipemiantes.Objetivo: Estudiar el efecto de la atorvastatina sobre la expresión deFas en tejido cerebral isquémico.Material y métodos: Quince ratas Sprague Dawley, machos, fuerondistribuidas en tres grupos (n = 5): un grupo control sin isquemia cerebral ydos experimentales (1 y 2) sometidos a isquemia cerebral global inducidapor paro respiratorio con D-tubocurarina. El grupo experimental 2 recibióatorvastatina durante las dos semanas previas a la inducción de isquemia(10 mg/kg/día, v.o.). Se realizó inmunohistoquímica por el sistema Avidina-Biotina-Peroxidasa a muestras de la corteza cerebral frontal.Resultados: Fas se expresó en el 38% de las células del tejido noisquémico. Esta expresión aumentó en el tejido cerebral isquémico a63% (P = 0,0003). La atorvastatina inhibió significativamente la expresiónde Fas en tejido cerebral isquémico en un 23% (P = 0,0007).Conclusiones: La atorvastatina disminuyó la expresión de Fas enisquemia cerebral, contribuyendo posiblemente a una inhibición de laapoptosis. Este mecanismo de acción pudiera explicar parte de su efectoneuroprotector. Estos hallazgos sugieren que la atorvastatina pudieraresultar beneficiosa en la prevención primaria de los ataques isquémicos

Background: The physiopathology of the brain ischemia includes many events such as important apoptotic mechanisms were Fas protein is expressed. Statins have neuroprotector effects that are independent of their blood lipid reducing actions. Objective: Study of the atorvastatin effect on Fas expression in brain ischemia. Material and methods: Fifteen male Sprague Dawley rats were distributed in three groups (n=5), the control group without cerebral ischemia, in the other two experimental groups (1 and 2) a global cerebral ischemia were induced by respiratory arrest (D-tubocurarin). Experimental group 2 was treated with atorvastatin (10 mg/Kg/d, p.o.) during two weeks before the induction of the ischemia. Inmunohistochemical study of brain frontal specimens was done by the Avidin- Biotin-Peroxidase method. Results: Fas was expressed in 38% of cells in non ischemic brain, this expression increased to 63% of cells in inchemic brain samples (P = 0.0003) Fas expression in ischemia was significantly inhibited by atorvastatin in 23% (P = 0.0007) Conclusions: Atorvastatin decreased Fas expression in ischemic brain. This fact must be taken into account to explain the atorvastatin neuroprotection and suggests the potential use such as primary prevention for ischemic attacks

Functionality of nitrated acetylcholine receptor: the two-step formation of nitrotyrosines reveals their differential role in effectors binding.

The presence of nitrotyrosines is associated with several neurodegenerative pathologies. We evaluated the functionality of the nicotinic acetylcholine receptor possessing nitrotyrosines. The spectrum of the nitrated receptor displays an absorption band characteristic of ortho-nitrophenol. The presence of carbamylcholine in the agonist site prevented the effect of nitration by tetranitromethane in some conditions. The nitration occurred with two discrete steps and pointed out the differential involvement of tyrosines in the binding of acetylcholine and neurotoxin. We concluded that at least two residues involved in agonist binding can be nitrated, which bring similar contributions to the binding energy of the neurotransmitter.

Iodo-alpha-conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors.

The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [(3)H]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the alpha/delta interface. An analogue of alpha-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the alpha/delta subunit interface. Alpha-conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin MI binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [(125)I]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-1)); k(on) is 8.5 x 10(7) min(-1) M(-1). The calculated k(d) is 0.98 nM. This potency is approximately 20-fold higher than the unmodified alpha-MI peptide. Unlike [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.

Toward atomic-scale understanding of ligand recognition in the muscle nicotinic receptor.

The nicotinic receptor at the motor endplate has served as a prototype for understanding structure, function and ligand recognition in the superfamily of pentameric ligand-gated ion channels. Yet despite this advanced state of knowledge, atomic-scale understanding of such elementary processes as ligand recognition has remained elusive owing to the lack of a high-resolution x-ray structure. However, the field has recently entered a state of rapid advancement following the discovery and atomic structural determination of the water-soluble acetylcholine binding protein (AChBP), a homolog of the receptor ligand binding domain. The AChBP structure provides the theoretical foundation for generating homology models of the corresponding receptor ligand binding domains within this structural family of receptors. Experimental assignment of residue equivalence between AChBP and receptor subunits subsequently yielded homology models ready for experimental testing. One such test is computational determination of ligand docking orientation in conjunction with mutagenesis of predicted contact residues and measurements of ligand binding affinity. Applied to different analogs of the competitive antagonist curare, docking computations that incorporate intrinsic protein flexibility reveal fundamentally distinct orientations of each analog bound to AChBP. The different contact residues predicted for each analog were tested and confirmed by mutagenesis of AChBP followed by measurements of ligand binding. By applying the same computational and experimental approaches to the adult human muscle AChR, we find that the two curare analogs also dock in distinctly different orientations. Thus subtle structural changes in the ligand, and by extension, structural differences in non-conserved residues among receptor subtypes and species, can dramatically alter the orientation of the bound ligand. The results have important implications for design of drugs targeting nicotinic receptors and members of the superfamily of pentameric ligand-gated ion channels.

[Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor]. / Chuvstvitel'nyi neradioaktivnyi metod skrininga soedinenii, vzaimodeistvuiushchikh s neironnym alpha7-kholinoretseptorom.

A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).

Interactions of cationic drugs and cardiac glycosides at the hepatic uptake level: studies in the rat in vivo, isolated perfused rat liver, isolated rat hepatocytes and oocytes expressing oatp2.

This paper deals with a crucial mechanism for interaction of basic drugs and cardiac glycosides at the hepatic uptake level. Available literature data is provided and new material is presented to picture the differential transport inhibition of bulky (type2) cationic drugs by a number of cardiac glycosides in rat liver. It is shown that the so called organic anion transporting peptide 2 (oatp2) is the likely interaction site: differential inhibition patterns as observed in oocytes expressing oatp2, could be clearly identified also in isolated rat hepatocytes, isolated perfused rat liver and the rat in vivo. The anticipation of transport interactions at the hepatic clearance level should be based on data on the relative affinities of interacting substrates for the transport systems involved along with knowledge on the pharmacokinetics of these agents as well as the chosen dose regimen in the studied species. This review highlights the importance of multispecific tranporter systems such as OATP, accommodating a broad spectrum of organic compounds of various charge, implying potential transport interactions that can affect body distribution and organ clearance.

Interaction of Drosophila acetylcholinesterases with D-tubocurarine: an explanation of the activation by an inhibitor.

Homotropic cooperativity in Drosophila melanogaster acetylcholinesterase seems to be a consequence of an initial substrate binding to a high-affinity peripheral substrate binding site situated around the negative charge of D413 (G335, Torpedo numbering). An appropriate mutation which turns the peripheral binding site to a low-affinity spot abolishes apparent activation but improves the overall enzyme effectiveness. This contradiction can be explained as less effective inhibition due to a shorter occupation of such a peripheral site. A similar effect can be achieved by an appropriate peripheral inhibitor such as TC, which can in special cases, when less effective heterotropic inhibition prevails over homotropic, acts as an activator. At the highest substrate concentrations, however, these enzymes are always inhibited, although steric components may influence the strength of inhibition like in the F368G mutant (F290, Torpedo numbering). Cooperative effects thus may include a steric component, but covering of the entrance must affect influx and efflux to different extents.

Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function.

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.

Influence of muscarinic ligands on the amplitudes of the evoked surface potential's late components in the optic tectum of the urodele Plethodon jordani.

Recordings of field potentials from the tectal surface of an urodele amphibian were obtained in an in vitro preparation under influence of various muscarinic drugs. Bath applied acetylcholine (ACh) led to no change in the amplitudes or the shape of the evoked potentials. If the ACh-esterase blocker (-)-physostigmine was applied synchronously, the late components of the surface potential increased in amplitude. The non-selective cholinergic agonist carbachol showed a similar effect which was partially diminished by the nicotinic antagonist d-tubocurarine chloride (d-TC) and the muscarinic antagonist atropine sulfate. The application of the non-selective muscarinic agonist (+)-pilocarpine hydrochloride led to an increase of the late oligo- and polysynaptic events. This effect was reduced by the M(1)-antagonist pirenzepine dihydrochloride. The presented findings suggest that muscarinic receptors play a more important role in tectal processing than assumed in previous studies which emphasized the role of nicotinic receptors.

Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.

Molecular determinants of (+)-tubocurarine binding at recombinant 5-hydroxytryptamine3A receptor subunits.

The 5-hydroxytryptamine type 3 (5-HT3) receptor is a transmitter-gated ion channel mediating neuronal excitation. The receptor native to neurons, or as a homopentameric assembly of 5-HT3A receptor subunits, displays a species-dependent pharmacology exemplified by a 1800-fold difference in the potency of (+)-tubocurarine [(+)-Tc] as an antagonist of the current response mediated by mouse and human receptor orthologs. Here, we attempt to identify amino acid residues involved in binding (+)-Tc by use of chimeric and mutant 5-HT3A subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5-HT3A (m5-HT3A) subunit by that of the human ortholog and vice versa exchanged the differential potency of (+)-Tc, demonstrating the ligand binding site to be contained wholly within this region. Mutagenesis of multiple amino acid residues within a putative binding domain that exchanged nonconserved residues between mouse and human receptors shifted the apparent affinity of (+)-Tc in a reciprocal manner. The magnitude of the shift increased with the number of residues (3, 5, or 7) exchanged, with septuple mutations of m5-HT3A and human 5-HT3A subunits producing a 161-fold decrease and 53-fold increase in the apparent affinity of (+)-Tc, respectively. The effect of point mutations was generally modest, the exception being m5-HT3A D206E, which produced a 9-fold decrease in apparent affinity. We conclude that multiple amino acids within a binding loop of human and mouse 5-HT3A subunits influence the potency of (+)-Tc.

Differential surface accessibility of alpha(187-199) in the Torpedo acetylcholine receptor alpha subunits.

We have probed the surface accessibility of residues alpha187 to alpha199 of the Torpedo acetylcholine receptor with monoclonal antibody 383C, which binds uniquely to these residues. However, 383C binds to only one of the two alpha subunits in the membrane-bound receptor, neither of the two subunits in carbamylcholine-desensitized receptor, and to both alpha subunits in Triton X-100 solubilized receptor. The kinetics of association and dissoci-ation of 383C with the peptide alpha(183-199) compared to those with the membrane-bound receptor suggest that all but a single hydrogen bond of affinity derives from contacts between this peptide and the monoclonal antibody paratope. Inhibition of 383C binding by alpha-bungarotoxin selectively directed to the alpha subunit correlated with the high-affinity d-tubocurarine binding site, along with a lack of inhibition by alpha-bungarotoxin directed to the alpha subunit correlated with the low-affinity d-tubocurarine binding site, suggests that the 383C epitope on the membrane-bound receptor resides on the alpha subunit associated with the high-affinity d-tubocurarine binding site. The results presented here suggest a structural basis for the differences between the two receptor acetylcholine binding sites.

Interaction of D-tubocurarine analogs with the 5HT3 receptor.

D-Tubocurarine is a potent competitive antagonist of two members of the ligand-gated ion channel family, the muscle-type nicotinic acetylcholine receptor (AChR) and serotonin type-3 receptor (5HT3R). We have used a series of analogs of D-tubocurarine to determine the effects of methylation, stereoisomerization and halogenation on the interaction of D-tubocurarine with the 5HT3R. The affinities of the analogs for the 5HT3R span a 200-fold concentration range and fall into three broad groups. The first group, with affinity constants (Ki) < 150 nM, consists of D-tubocurarine and analogs modified at the nitrogens or 7' hydroxyl. The fact that these compounds all have high affinity for the 5HT3R suggests that these portions of the ligand do not make interactions with the receptor that are critical for high-affinity binding. The second group, with Ki's in the 1-5 microM range, consists of analogs modified at the 12'-hydroxyl or the adjacent 13'-carbon, which suggests that this portion of the ligand makes interactions that are important for high-affinity binding. The third, very low affinity, group is a compound with altered stereoconfiguration at the 1 carbon, demonstrating the importance of proper configuration of the antagonist in ligand-receptor interactions. For the most part, this pattern of selectivity is similar to that for the AChR, suggesting that the structures of the ligand-binding sites of these two receptors share common structural features.