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Double seropositive myasthenia gravis (DSP-MG) has antibodies positive to both acetylcholine receptor (AChR) antibody and muscle-specific tyrosine kinase (MuSK) antibody. This study aims to delineate the clinical phenotype of DSP-MG and assess the histopathological correlation with thymoma. This is a retrospective case series conducted at a tertiary care hospital in South India between February 2018 and October 2021. We had 13 DSP-MG patients (seven females, mean age 60.77+/-14.24). The presentation was generalized in nine patients, bulbar in three, and ocular in one patient. Multi-detector computed tomography done in 11 patients showed thymoma in five and thymic hyperplasia in one. Four patients underwent thymectomy; histopathology showed Type A, B2 (n = 2), and AB thymomas. All 13 patients improved with anticholinesterases. Nine patients were administered immunosuppressants, three patients were given intravenous immunoglobulin, and a single patient underwent plasmapheresis. Our study shows that DSP-MG is more similar to the clinical phenotype of AChR-MG. Keywords: Acetylcholine receptor antibody, double positive myasthenia, MuSK receptor antibody, myasthenia gravis, thymoma, Author(s): Chirag S. Lalwani [1]; Hamsini Movva [1]; Sudheeran Kannoth (corresponding author) [1,2]; Vivek Nambiar [1]; Siby Gopinath [1]; Anandkumar Anandakuttan [1]; Saraf U. Umesh [1]; Sreelakshmi Narayanan [1]; Bijoy [...]

Systemic inflammatory response syndrome plays an important role in the development of sepsis. GABAergic and cholinergic pathways activation are considered important for inflammatory response regulation. Tumor necrosis factor (TNF)-[alpha], interleukin (IL)-1[beta], IL-12, IL-10, as well as inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) are important inflammatory mediators involved in the pathogenesis of sepsis. Muscimol, an active compound from the mushroom Amanita muscaria (L.) Lam., is a potent [GABA.sub.A] agonist, inhibits inflammatory response via activating [GABA.sub.A] receptor and vagus nerve. However, the effect of muscimol on lipopolysaccharide (LPS)-induced systemic inflammatory response is still unclear. Therefore, we studied the effects of muscimol on systemic inflammatory response and survival rate in endotoxemic mice. Mice endotoxemia was induced by LPS. Muscimol was given to mice or RAW264.7 cells 30 min before LPS (10 mg/kg, i.p., or 10 ng/mL, respectively). Mice received GABAergic and cholinergic receptor antagonists 30 min before muscimol and LPS. Muscimol decreased TNF- [alpha], IL-1[beta], IL-12, iNOS-derived NO, and increased IL-10 levels and survival rate after LPS treatment. Muscimol significantly decreased nuclear factor kappa B (NF-[kappa]B) activity, increased I[kappa]B expression, and decreased pIKK expression in LPS- treated RAW264.7 cells. GABAergic and cholinergic antagonists failed to reverse muscimol's protection in LPS-treated mice. In conclusion, muscimol protected against systemic inflammatory response in endotoxemic mice may be partially independent of GABAergic and cholinergic receptors. Key words: muscimol, systemic inflammatory response, LPS, GABA receptor, cholinergic receptor Le syndrome de reaction inflammatoire generalisee joue un role important dans le developpement de la septicemie. L'activation des voies de signalisation GABAergiques et cholinergiques est consideree comme etant importante dans la regulation de la reaction inflammatoire. Le facteur de necrose tumorale (TNF)-, l'interleukine (IL)-1[beta], l'IL-12, l'IL-10, de meme que l'oxyde nitrique (NO) derive de la NO synthase inductible (iNOS) sont d'importants mediateurs inflammatoires, qui jouent un role dans la pathogenese de la septicemie. Le muscimol, un compose actif issu du champignon Amanita muscaria, puissant agoniste des recepteurs GABAA, permet d'inhiber la reaction inflammatoire par l'intermediaire de l'activation des recepteurs GABAA et du nerf vague. Cependant, l'effet du muscimol sur la reaction inflammatoire generalisee engendree par les lipopolysaccharides (LPS) demeure a clarifier. Par consequent, nous avons etudie les effets du muscimol sur la reaction inflammatoire generalisee et sur le taux de survie chez la souris endotoxemique. Nous avons obtenu une endotoxemie chez des souris a l'aide de LPS. Nous avons administre du muscimol chez des souris ou chez des cellules RAW264.7 30 minutes avant les LPS (10 mg/kg, i.p., ou 10 ng/ml, respectivement). Les souris ont recu des antagonistes GABAergiques et cholinergiques 30 minutes avant le muscimol et les LPS. Apres l'administration des LPS, le muscimol a entraine une diminution des taux de TNF-[alpha], d'IL-1[beta], d'IL-12, de NO derive de l'iNOS, avec une diminution des taux d'IL-10 de meme que du taux de survie. Le muscimol entrainait une diminution marquee de l'activite du NF-[kappa]B, une augmentation de l'activite de I'I[kappa]B, ainsi qu'une diminution de l'expression de pIKK dans les cellules RAW264.7 exposees aux LPS. Les antagonistes GABAergiques et cholinergiques n'ont pas permis de contrer la protection qu'engendrait le muscimol chez les souris auxquelles des LPS etaient administres. En conclusion, la protection contre la reaction inflammatoire generalisee obtenue avec le muscimol chez des souris endotoxemiques pourrait etre partiellement independante des recepteurs GABAergiques et cholinergiques. [Traduit par la Redaction] Mots-cles : muscimol, reaction inflammatoire generalisee, LPS, recepteurs du GABA, recepteur cholinergique, Introduction Systemic inflammatory response syndrome (SIRS), mainly caused by Gram-negative bacteria, plays a vital role in the development of sepsis (Kaukonen et al. 2015; Chakraborty and Burns 2021). It estimates [...]

The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD., Introduction Due to an aging population, it is predicted that by 2040, neurodegenerative diseases, such as Alzheimer's disease (AD), will be the second most common cause of morbidity in the [...]

Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in acetylcholine receptor (AChR) function. Disease-associated point mutants can reveal the unsuspected functional significance of mutated residues. We identified two pathogenic mutations in the extracellular domain of the AChR α subunit (AChR α) in a patient with myasthenic symptoms since birth: a V188M mutation in the C-loop and a heteroallelic G74C mutation in the main immunogenic region. The G74C mutation markedly reduced surface AChR expression in cultured cells, whereas the V188M mutant was expressed robustly but had severely impaired kinetics. Single-channel patch-clamp analysis indicated that V188M markedly decreased the apparent AChR channel opening rate and gating efficiency. Mutant cycle analysis of energetic coupling among conserved residues within or dispersed around the AChRα C-loop revealed that V188 is functionally linked to Y190 in the C-loop and to D200 in β-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in β-strand 7 with Y190 and with D200. Cumulatively, these results indicate that V188 of AChR α is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating., Introduction Congenital myasthenic syndromes (CMSs) are heterogeneous disorders in which the safety margin of neuromuscular transmission is impaired by one or more defects in motor endplate-associated proteins (1). Most defects [...]

The aim of this study is to investigate whether nicotinic acetylcholine receptors (nAChRs) are involved in the modulation of functional activity of late endothelial progenitor cells (EPCs) induced by nicotine. Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low- density lipoprotein (DiI-acLDL) uptake and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1) binding. Expression of von Willbrand factor (vWF), kinase insert domain receptor (KDR), and α7 nAChR was detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 32 h with either vehicle or nicotine with or without pre-incubation of nAChR antagonism, mecamylamine, or α-bungarotoxin. The viability, migration, and in vitro vasculogenesis activity of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with 10 nmol/L nicotine enhanced viable, migratory, adhesive, and in vitro vasculogenesis capacity of late EPCs. The effect of nicotine on late EPCs can be attenuated by mecamylamine or α-bungarotoxin. In conclusion, nicotine improves the functional activity of late EPCs via nAChRs. Key words: nicotinic acetylcholine receptor, late endothelial progenitor cells, nicotine, mecamylamine, α- bungarotoxin. O11-032(BCB10292, 2011-0030)Le but de cette etude etait de determiner si les recepteurs nicotiniques de l'acetylcholine (nAChR) sont impliques dans la modulation de l'activite fonctionnelle des cellules souches endotheliales (CSE) tardives induites par la nicotine. Les cellules mononucleaires (CMN) totales ont ete isolees de sang de cordon ombilical humain par centrifugation sur gradient de densite Ficoll, et elles ont ete ensemencees sur des plaques de culture recouvertes de fibronectine. Les CSE tardives etaient positives a la captation de lipoproteines de faible densite acetylees et marquees au 1,1- dioctadecyl-3,3,3,3-tetra methylindocarbocyanine (Dil-acLDL), et a la liaison de l'agglutinine d'Ulex europaeus conjuguee a l'isothyocyanate de fluoresceine (UEA-1). L'expression du facteur de von Willbrand (vWF), du recepteur KDR (kinase insert domain receptor) et du nAChR α7 a ete detectee par coloration indirecte en immunofluorescence. Les CSE tardives de passages 3 a 5 ont ete traitees pendant 32 heures avec un vehicule ou de la nicotine, avec ou sans preincubation avec un antagoniste du nAChR α7, la mecamylamine ou lα-bungarotoxine. La viabilite, la migration et la vasculogenese in vitro des CSE tardives ont ete examinees a l aide d un dosage au bromure de 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), d un test en chambre de Boyden modifie et d un test d angiogenese in vitro, respectivement. Le test d adhesion des CSE tardives a ete realise en repiquant les cellules sur des plaques de culture recouvertes de fibronectine, et les cellules adherentes ont ete comptees. Une incubation avec 10 nmol/L de nicotine augmentait la viabilite, la migration, l adhesion et la vasculogenese in vitro des CSE tardives. L effet de la nicotine sur les CSE tardives pouvait etre attenue par la mecamylamine ou l α-bungarotoxine. En conclusion, la nicotine ameliore l activite fonctionnelle des CSE tardives par l intermediaire des nAChR. Mots-cles : recepteur nicotinique de l'acetylcholine, cellules souches endotheliales tardives, nicotine, mecamylamine, α-bungarotoxine. [Traduit par la Redaction], Introduction Nicotine is an alkaloid compound found in the nightshade family of plants. Although nicotine exposure induces vascular endothelial dysfunction and thus impairs neovascularization, recent studies have shown that nicotine [...]

Pannexin-1 (Panx1) plays a role in the release of ATP and glutamate in neurons and astrocytes. Panx1 can be opened at the resting membrane potential by extracellular ATP via the [...]

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms. negative regulation | postsynaptic | presynaptic | retrograde signal | neurotransmitter doi/ 10.1073/pnas.1004956107

Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type [alpha][1.sub.2][beta][gamma][delta] and neuronal [alpha]3[beta]2 and [alpha]4[beta]2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities govemed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ([approximately equal to]2.4[Angstrom]) show toxins neatly imbedded within the nest of aromatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the sequence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity. acetylcholine binding protein | marine phycotoxins | nicotinic acetylcholine receptor | pharmacological and structural analyses | seafood poisoning doi/10.1073/pnas.0912372107

Efficient neurotransmission at chemical synapses relies on spatial congruence between the presynaptic active zone, where synaptic vesicles fuse, and the postsynaptic differentiation, where neurotransmitter receptors concentrate. Diverse molecular systems have [...]

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a [G.sub.i]-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current ([I.sub.f]) or L-type [Ca.sup.2+] currents ([I.sub.Ca,L]), respectively; and 2) direct [G.sub.i]-coupled activation of ACh-activated potassium current ([I.sub.KACh]). More recent studies, however, have indicated that [Ca.sup.2+] cycling by the sarcoplasmic reticulum within SANC (referred to as a [Ca.sup.2+] clock) generates rhythmic, spontaneous local [Ca.sup.2+] releases (LCR) that are AC-PKA dependent. LCRs activate [Na.sup.+]-[Ca.sup.2+] exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was ~10 nM, half maximal inhibition ([IC.sub.50]) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. Gi inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that If blockade did not affect BRR at any [CCh] and that [I.sub.KACh] activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At [IC.sub.50], CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by ~50%. The dose response of BRR to CCh in the presence of IKACh blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At [IC.sub.50], CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of [IC.sub.50] CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that [Ca.sup.2+] cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulationinduced BRR is entirely dependent on [G.sub.i] activation and the extent of [G.sub.i] coupling to [Ca.sup.2+] cycling via PKA signaling or to [I.sub.KACh]: at low [CCh], [I.sub.KACh] activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent [Ca.sup.2+] signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent [Ca.sup.2+] signaling and IKACh activation underlies a more pronounced BRR. submembrane [Ca.sup.2+] release; ion channels; protein kinase A phosphorylation; signal transduction

The enteric nervous system (ENS) controls gut function. P2X receptors and nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that mediate fast synaptic excitation in the ENS. Close molecular coupling in enteric neuronal membranes contributes to a mutually inhibitory interaction between these receptors; this effect is called cross-inhibition. We studied the molecular mechanisms responsible for cross-inhibition. Whole cell patch-clamp techniques were used to measure P2X-and nAChR-mediated currents in cultured enteric neurons and HEK-293 cells. In cultured myenteric neurons, ACh (3 mM) and ATP (1 mM) coapplication evoked an inward current that was only 57 [+ or -] 6% (P < 0.05) of the predicted current that would have occurred if the two populations of channels were activated independently. In HEK-293 cells coexpressing [[alpha].sub.3][[beta].sub.4] nAChR/[P2X.sub.2] receptors, coapplication of ATP and ACh caused a current that was 58 [+ or -] 7% of the predicted current (P < 0.05). To test the importance of P2X subunit COOH-temainal tail length on cross-inhibition, [P2X.sub.3] and [P2X.sub.4] subunits, which have shorter COOH-terminal tails, were studied. Cross-inhibition with [[alpha].sub.3][[beta].sub.4] nAChRs and [P2X.sub.3] or [P2X.sub.4] subunits was similar to that occurring with [P2X.sub.2] subunits. P2X receptor or [[alpha].sub.3][[beta].sub.4] nAChR desensitization did not prevent receptor cross-inhibition. These data indicate that the [[alpha].sub.3][[beta].sub.4]-P2X receptor interaction is not restricted to [P2X.sub.2] subunits. In addition, active and desensitized conformations of the P2X receptor inhibit nAChR function. These molecular interactions may modulate the function of synapses that use ATP and ACh as fast synaptic transmitters in the ENS. enteric nervous system; synaptic transmission; ligand-gated ion channels

In the course of synaptic transmission in the brain and periphery, acetylcholine receptors (AChRs) rapidly transduce a chemical signal into an electrical impulse. The speed of transduction is facilitated by [...]

The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs ([alpha]S269I, [[beta]V266M, [epsilon]L221F, [epsilon]T264P, and [epsilon]L269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel-based approach to estimate the contribution of [Ca.sup.2+] to the total cation currents, we also found that none of these mutants affects the [Ca.sup.2+]-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the [Ca.sup.2+]-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM [Na.sup.+], 1.8 mM [Ca.sup.2+], and 1.7 mM [Mg.sup.2+] on the extracellular side of cell-attached patches turned out be in the 5.0-9.4 pS range, representing a fractional [Ca.sup.2+] current of ~14%, on average. Remarkably, these values are nearly identical to those we estimated for the NR1-NR2A N-methyl-D-aspartate receptor (NMDAR), which has generally been considered to be the main neurotransmitter-gated pathway of [Ca.sup.2+] entry into the cell. Our estimate of the rat NMDAR [Ca.sup.2+] conductance (using the same single-channel approach as for the AChR but in the nominal absence of extracellular [Mg.sup.2+]) was 7.9 pS, corresponding to a fractional [Ca.sup.2+] current of 13%.

We estimated the unliganded opening and closing rate constants of neuromuscular acetylcholine receptor-channels (AChRs) having mutations that increased the gating equilibrium constant. For some mutant combinations, spontaneous openings occurred in clusters. For 25 different constructs, the unliganded gating equilibrium constant ([E.sub.0]) was correlated with the product of the predicted fold-increase in the diliganded gating equilibrium constant caused by each mutation alone. We estimate that (i) [E.sub.0] for mouse, wild-type [[alpha].sub.2][beta][delta][epsilon] AChRs is [approximately equal to] 1.15 x [10.sup-7]; (ii) unliganded AChRs open for [approximately equal to] 80 [micro]s, once every [approximately equal to] 15 min; (iii) the affinity for ACh of the O(pen) conformation is [approximately equal to] 10 nM, or [approximately equal to] 15,600 times greater than for the C(losed) conformation; (iv) the ACh-monoliganded gating equilibrium constant is [approximately equal to] 1.7 x [10.sup.-3]; (v) the C [ right arrow] O isomerization reduces substantially ACh dissociation, but only slightly increases association; and (vi) ACh provides only [approximately equal to] 0.9 [k.sub.B]T more binding energy per site than carbamylcholine but [approximately equal to] 3.1 [k.sub.B]T more than choline, mainly because of a low O conformation affinity. Most mutations of binding site residue [alpha]W149 increase [E.sub.0]. We estimate that the mutation [alpha]W149F reduces the ACh affinity of C only by 13-fold, but of O by 190-fold. Rate--equilibrium free-energy relationships for different regions of the protein show similar slopes ([phi] values) for un- vs. diliganded gating, which suggests that the conformational pathway of the gating structural change is fundamentally the same with and without agonists. Agonist binding is a perturbation that (like most mutations) changes the energy, but not the mechanism, of the gating conformational change. allosteric | protein | synapse | kinetics | spontaneous

Wnt proteins regulate the formation of central synapses by stimulating synaptic assembly, but their role at the vertebrate neuromuscular junction (NMJ) is unclear. Wnt3 is expressed by lateral motoneurons of the spinal cord during the period of motoneuron-muscle innervation. Using gain- and loss-of-function studies in the chick wing, we demonstrate that Wnt signaling is necessary for the formation of acetylcholine receptor (AChR) clusters without affecting muscle growth. Similarly, diaphragms from Dishevelled-1 mutant mice with deficiency in Wnt signaling exhibit defects in cluster distribution. In cultured myotubes, Wnt3 increases the number and size of AChR clusters induced by agrin, a nerve-derived signal critical for NMJ development. Wnt3 does not signal through the canonical Wnt pathway to induce cluster formation. Instead, Wnt3 induces the rapid formation of unstable AChR micro-clusters through activation of Rac1, which aggregate into large clusters only in the presence of agrin. Our data reveal a role for Wnts in post-synaptic assembly at the vertebrate NMJ by enhancing agrin function through Rac1 activation. acetylcholine receptor | clustering | Dvl1 mutant | neuromuscular junction | Rac