Protein Tyrosine Phosphatase Receptor Type R (PTPRR) Reduces AChR Clustering by Dephosphorylating MuSK.

Neuromuscular junction (NMJ) formation and maintenance depend on the proper localization and concentration of various molecules at synaptic contact sites. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane is a cardinal event in NMJ formation. Muscle-specific tyrosine kinase (MuSK), which functions depending on its phosphorylation, plays an essential role in AChR clustering. In the present study, we used plasmid-based biochemical screening and determined that protein tyrosine phosphatase receptor type R (PTPRR) is responsible for dephosphorylating MuSK on tyrosine residue 754. Furthermore, we showed that PTPRR significantly reduced MuSK-dependent AChR clustering in C2C12 myotubes. Collectively, these data illustrate a negative regulation function of PTPRR in AChR clustering.

Ancestral acetylcholine receptor ß-subunit forms homopentamers that prime before opening spontaneously.

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the ß-, δ-, and ε-subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here, we discover that a reconstructed ancestral muscle-type ß-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings attest to the homopentameric origin of the muscle-type acetylcholine receptor, and demonstrate that signature features of its function are both independent of agonist and do not necessitate the complex heteropentameric architecture of the modern-day protein.

New Coumarin Derivatives as Cholinergic and Cannabinoid System Modulators.

In the last years, the connection between the endocannabinoid system (eCS) and neuroprotection has been discovered, and evidence indicates that eCS signaling is involved in the regulation of cognitive processes and in the pathophysiology of Alzheimer's disease (AD). Accordingly, pharmacotherapy targeting eCS could represent a valuable contribution in fighting a multifaceted disease such as AD, opening a new perspective for the development of active agents with multitarget potential. In this paper, a series of coumarin-based carbamic and amide derivatives were designed and synthesized as multipotent compounds acting on cholinergic system and eCS-related targets. Indeed, they were tested with appropriate enzymatic assays on acetyl and butyryl-cholinesterases and on fatty acid amide hydrolase (FAAH), and also evaluated as cannabinoid receptor (CB1 and CB2) ligands. Moreover, their ability to reduce the self-aggregation of beta amyloid protein (Aß42) was assessed. Compounds 2 and 3, bearing a carbamate function, emerged as promising inhibitors of hAChE, hBuChE, FAAH and Aß42 self-aggregation, albeit with moderate potencies, while the amide 6 also appears a promising CB1/CB2 receptors ligand. These data prove for the new compounds an encouraging multitarget profile, deserving further evaluation.

In Vivo Receptor Visualization and Evaluation of Receptor Occupancy with Positron Emission Tomography.

Positron emission tomography (PET) is useful for noninvasive in vivo visualization of disease-related receptors, for evaluation of receptor occupancy to determine an appropriate drug dosage, and for proof-of-concept of drug candidates in translational research. For these purposes, the specificity of the PET tracer for the target receptor is critical. Here, we review work in this area, focusing on the chemical structures of reported PET tracers, their Ki/Kd values, and the physical properties relevant to target receptor selectivity. Among these physical properties, such as cLogP, cLogD, molecular weight, topological polar surface area, number of hydrogen bond donors, and pKa, we focus especially on LogD and LogP as important physical properties that can be easily compared across a range of studies. We discuss the success of PET tracers in evaluating receptor occupancy and consider likely future developments in the field.

The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms.

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.

Rare slow channel congenital myasthenic syndromes without repetitive compound muscle action potential and dramatic response to low dose fluoxetine.

Congenital myasthenic syndromes are rare hereditary disorders caused by mutations associated with proteins of the neuromuscular junction. Abnormal ''gain of function'' mutations result in prolonged nicotinic acetylcholine receptor channel open state causing a rare subtype of CMS, slow-channel CMS (SCCMS). Mutations in the delta subunit encoding the gene, CHRND, resulting in SCCMS are extremely rare. An important clue to the diagnosis of SCCMS is repetitive CMAP's. Fluoxetine, usually at high doses, is used to treat SCCMS. The mutation, recently described in one patient, was identified by whole exome sequencing and validated, and its segregation with the disease was ascertained by Sanger sequencing. Here, we describe clinical and genetic findings of an early onset SCCMS patient carrying a very rare missense mutation c.880C > T in CHRND causing a highly conserved leucine to phenylalanine substitution in the M2 domain of CHRND. The patient had no repetitive CMAP. He had a dramatic response to fluoxetine at low-moderate doses (40 mg/day), increasing over months: Being wheelchair bound, he could walk independently after treatment. Rare cases may offer insight into the pathological gating mechanism leading to CMS. SCCMS should be suspected even without a repetitive CMAP. Fluoxetine at relatively low doses can be a very effective treatment.

Affinity maturation is required for pathogenic monovalent IgG4 autoantibody development in myasthenia gravis.

Pathogenic muscle-specific tyrosine kinase (MuSK)-specific IgG4 autoantibodies in autoimmune myasthenia gravis (MG) are functionally monovalent as a result of Fab-arm exchange. The development of these unique autoantibodies is not well understood. We examined MG patient-derived monoclonal autoantibodies (mAbs), their corresponding germline-encoded unmutated common ancestors (UCAs), and monovalent antigen-binding fragments (Fabs) to investigate how affinity maturation contributes to binding and immunopathology. Mature mAbs, UCA mAbs, and mature monovalent Fabs bound to MuSK and demonstrated pathogenic capacity. However, monovalent UCA Fabs bound to MuSK but did not have measurable pathogenic capacity. Affinity of the UCA Fabs for MuSK was 100-fold lower than the subnanomolar affinity of the mature Fabs. Crystal structures of two Fabs revealed how mutations acquired during affinity maturation may contribute to increased MuSK-binding affinity. These findings indicate that the autoantigen drives autoimmunity in MuSK MG through the accumulation of somatic mutations such that monovalent IgG4 Fab-arm-exchanged autoantibodies reach a high-affinity threshold required for pathogenic capacity.

Acetylcholine and Acetylcholine Receptors: Textbook Knowledge and New Data.

It was a pleasure to receive a proposal to organize and be a guest editor of a Special Issue ofBiomolecules. This is the field in which I am working and personally know some of the leadingscientists. My narrow field is the research on the peptide and protein neurotoxins from animalvenoms and their application as sophisticated tools for analysis of nicotinic acetylcholine receptors(nAChRs) [...].

Dynamic heterogeneity and non-Gaussian statistics for ganglioside GM1s and acetylcholine receptors on live cell membrane.

We have carried out a comparative study of the lateral motion of ganglioside GM1, which is a glycosphingolipid residing on the outer leaflet of the plasma membrane, and acetylcholine receptor (AChR), which is a well-characterized ion channel. Both the lipid molecules and the transmembrane proteins reside on the plasma membranes of live Xenopus muscle cells. From a thorough analysis of a large volume of individual molecular trajectories obtained from more than 300 live cells over a wide range of sampling rates and long durations, we find that the GM1s and AChRs share the same dynamic heterogeneity and non-Gaussian statistics. Our measurements with the ATP-depleted cells reveal that the diffusion dynamics of the GM1s and AChRs is uniformly affected by the intracellular ATP level of the living muscle cells, further demonstrating that membrane diffusion is strongly coupled to the dynamics of the underlying cortical actin network, as predicted by the dynamic picket-fence model.

Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs.

At vertebrate neuromuscular junctions (NMJs), the synaptic basal lamina contains different extracellular matrix (ECM) proteins and synaptogenic factors that induce and maintain synaptic specializations. Here, we report that podosome-like structures (PLSs) induced by ubiquitous ECM proteins regulate the formation and remodeling of acetylcholine receptor (AChR) clusters via focal ECM degradation. Mechanistically, ECM degradation is mediated by PLS-directed trafficking and surface insertion of membrane-type 1 matrix metalloproteinase (MT1-MMP) to AChR clusters through microtubule-capturing mechanisms. Upon synaptic induction, MT1-MMP plays a crucial role in the recruitment of aneural AChR clusters for the assembly of postsynaptic specializations. Lastly, the structural defects of NMJs in embryonic MT1-MMP-/- mice further demonstrate the physiological role of MT1-MMP in normal NMJ development. Collectively, this study suggests that postsynaptic MT1-MMP serves as a molecular switch to synaptogenesis by modulating local ECM environment for the deposition of synaptogenic signals that regulate postsynaptic differentiation at developing NMJs.

Voluntary movement relies on skeletal muscle cells and nerve cells being able to communicate with one another. This communication occurs at a specialized region called the neuromuscular junction, or NMJ for short. These junctions are surrounded by a meshwork of proteins, known as the matrix, which structurally supports the nerve and muscle cells. Muscle cells contain proteins called acetylcholine receptors on their cell surface. When these receptors cluster together at the NMJ, this allows nerve cells to communicate with the muscle cell and tell the muscle to contract. However, these clusters can also form spontaneously without the help of nerve cells at regions away from the communication site. Alongside these spontaneous clusters of acetylcholine receptors are dynamic actin-enriched structures. These structures are responsible for releasing enzymes that digest the surrounding matrix and are commonly found in migrating cells. But as skeletal muscle cells do not migrate, it remained unclear what purpose these structures serve at the NMJ. Now, Chan et al. have used advanced microscopy techniques to show how these actin-enriched structures can help acetylcholine receptors cluster together at the site of communication between the nerve and muscle cells. The experiments showed that these structures direct a molecule called MT1-MMP to the muscle surface. This molecule then clears the surrounding matrix so that signals sent from the nerve can be effectively deposited at the narrow space between these two cells. When the muscle cells receive this initiating signal, acetylcholine receptors are recruited from the spontaneously formed clusters to the communication site, allowing the muscle to contract. When MT1-MMP was experimentally eliminated in mice, this disrupted the recruitment of acetylcholine receptors to the NMJ. Overall, these experiments help researchers understand how clearing the matrix between nerve and muscle cells contributes to the deposition of factors that build the communication site at developing NMJs. In the future this might help develop treatments for movement disorders caused by abnormalities that affect the clearing of matrix proteins in these junctions.

Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure.

MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with ß2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.

Binding of Sulfoxaflor to Aplysia californica-AChBP: Computational Insights from Multiscale Approaches.

Structural features and binding properties of sulfoxaflor (SFX) with Ac-AChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics, and QM/QM'). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM' calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules-mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH···π and π···π interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine-based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.

Neural anti-inflammatory action mediated by two types of acetylcholine receptors in the small intestine.

Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.

Efficiency measures the conversion of agonist binding energy into receptor conformational change.

Receptors alternate between resting↔active conformations that bind agonists with low↔high affinity. Here, we define a new agonist attribute, energy efficiency (η), as the fraction of ligand-binding energy converted into the mechanical work of the activation conformational change. η depends only on the resting/active agonist-binding energy ratio. In a plot of activation energy versus binding energy (an "efficiency" plot), the slope gives η and the y intercept gives the receptor's intrinsic activation energy (without agonists; ΔG0). We used single-channel electrophysiology to estimate η for eight different agonists and ΔG0 in human endplate acetylcholine receptors (AChRs). From published equilibrium constants, we also estimated η for agonists of KCa1.1 (BK channels) and muscarinic, γ-aminobutyric acid, glutamate, glycine, and aryl-hydrocarbon receptors, and ΔG0 for all of these except KCa1.1. Regarding AChRs, η is 48-56% for agonists related structurally to acetylcholine but is only ∼39% for agonists related to epibatidine; ΔG0 is 8.4 kcal/mol in adult and 9.6 kcal/mol in fetal receptors. Efficiency plots for all of the above receptors are approximately linear, with η values between 12% and 57% and ΔG0 values between 2 and 12 kcal/mol. Efficiency appears to be a general attribute of agonist action at receptor binding sites that is useful for understanding binding mechanisms, categorizing agonists, and estimating concentration-response relationships.

Molecular and pharmacological characterization of an acetylcholine-gated chloride channel (ACC-2) from the parasitic nematode Haemonchus contortus.

Nematode cys-loop ligand-gated ion channels (LGICs) have been shown to be attractive targets for the development of novel anti-parasitic drugs. The ACC-1 family of receptors are a unique group of acetylcholine-gated chloride channels present only in invertebrates, and sequence analysis suggests that they contain a novel binding site for acetylcholine. We have isolated a novel member of this family, Hco-ACC-2, from the parasitic nematode Haemonchus contortus and using site-directed mutagenesis, electrophysiology and molecular modelling examined how two aromatic amino acids in the binding site contributed to agonist recognition. It was found that instead of a tryptophan residue in binding loop B, which essential for ligand binding in mammalian nAChRs, there is a phenylalanine (F200) in Hco-ACC-2. Amino acid changes at F200 to either a tyrosine or tryptophan were fairly well tolerated, where a F200Y mutation resulted in a channel hypersensitive to ACh and nicotine as well as other cholinergic agonists such as carbachol and methacholine. In addition, both pyrantel and levamisole were partial agonists at the wild-type receptor and like the other agonists showed an increase in sensitivity at F200Y. On the other hand, in Hco-ACC-2 there is a tryptophan residue at position 248 in loop C that appears to be essential for receptor function, as mutations to either phenylalanine or tyrosine resulted in a marked decrease in agonist sensitivity. Moreover, mutations that swapped the residues F200 and W248 (ie. F200W/W248F) produced non-functional receptors. Overall, Hco-ACC-2 appears to have a novel cholinergic binding site that could have implications for the design of specific anthelmintics that target this family of receptors in parasitic nematodes.

Modified neonicotinoid insecticide with bi-directional selective toxicity and drug resistance.

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model was established based on the molecular structures and the negative logarithm of experimental lethal concentration 50 values (pLC50) of neonicotinoid insecticides. Then, the mechanisms of bi-directional selective toxic effects and drug resistance were determined using homology modeling and molecular docking analyses. The results of the model showed that the 1-, 2-, 4-, and 12- positions of neonicotinoid insecticides strongly affected their toxicity, and that the introduction of bulky or electropositive groups at these positions could increase the pLC50 values. Using Compound 19 as a template, we designed 37 derivatives with greater toxicity (increased by 0.04-11.45%). Among them, 20 derivatives had bioconcentrations lower than that of Compound 19 (reduced by 0.38-147.88%). Further screening of Compound 19 and the 20 derivatives mentioned above by homology modeling and acetylcholine receptors (AChRs) molecular docking analyses showed that 10 derivatives had bi-directional selective toxic effects against pests and bees. Further docking analyses of Compound 19 and these 10 derivatives identified that Derivative-33 showed decreased docking with superoxide dismutase (SOD) and glutathione S transferase (GST) in pests and enhanced docking with these enzymes in bees, indicating bi-directional selective resistance for pests and bees. Accordingly, Derivative-33 was selected as a new insecticide with high toxicity to pests and low toxicity to bees (bi-directional selective toxicity), low resistance in pest populations, and high resistance in bee populations. This study provides valuable reference data and will be useful for the development of strategies to produce new environmentally friendly pesticides.

Immunostick ELISA for rapid and easy diagnosis of myasthenia gravis.

A rapid and simple immunostick immunosorbent ELISA has been developed for the diagnosis of myasthenia gravis. It is based on coating immunosticks with the major myasthenia gravis autoantigens acetylcholine receptor (AChR) or MuSK and it can be performed in <1 h, in a non-specialized examination place by non-skilled personnel. The resulting chromogen precipitate on the sticks, evaluated by eye, was proved a reliable indication of the presence of autoantigen-specific autoantibodies. Although this assay is mostly qualitative, compared to the results obtained from the gold-standard radioimmunoprecipitation assays, its specificity for anti-AChR antibodies was 99.1% (102/103 negative sera) and its sensitivity was 91.1% (72/79) or 97.5% (77/79) depending on counting the 5 ambiguous sera as negative or positive, respectively. Smaller scale experiments suggested that the method is efficient for anti-MuSK antibodies, and may also be used with whole blood instead of serum.

Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a 'model hopping' approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

Full and partial agonists evoke distinct structural changes in opening the muscle acetylcholine receptor channel.

The muscle acetylcholine (ACh) receptor transduces a chemical into an electrical signal, but the efficiency of transduction, or efficacy, depends on the particular agonist. It is often presumed that full and partial agonists elicit the same structural changes after occupancy of their binding sites but with differing speed and efficiency. In this study, we tested the alternative hypothesis that full and partial agonists elicit distinct structural changes. To probe structural changes, we substituted cysteines for pairs of residues that are juxtaposed in the three-dimensional structure and recorded agonist-elicited single-channel currents before and after the addition of an oxidizing reagent. The results revealed multiple cysteine pairs for which agonist-elicited channel opening changes after oxidative cross-linking. Moreover, we found that the identity of the agonist determined whether cross-linking affects channel opening. For the αD97C/αY127C pair at the principal face of the subunit, cross-linking markedly suppressed channel opening by full but not partial agonists. Conversely, for the αD97C/αK125C pair, cross-linking impaired channel opening by the weak agonist choline but not other full or partial agonists. For the αT51C/αK125C pair, cross-linking enhanced channel opening by the full agonist ACh but not other full or partial agonists. At the complementary face of the subunit, cross-linking between pairs within the same ß hairpin suppressed channel opening by ACh, whereas cross-linking between pairs from adjacent ß hairpins was without effect for all agonists. In each case, the effects of cross-linking were reversed after addition of a reducing reagent, and receptors with single cysteine substitutions remained unaltered after addition of either oxidizing or reducing reagents. These findings show that, in the course of opening the receptor channel, different agonists elicit distinct structural changes.