Affinity For Acetylcholine Research Articles

Impact of a worker bee thoracic ganglion RIC-3 variant on the actions of acetylcholine and neonicotinoids on nicotinic receptors in Apis mellifera.

A transmembrane thioredoxin (TMX3) enables the functional expression of insect nicotinic acetylcholine receptors (nAChRs) in Xenopus laevis oocytes, while co-factors RIC-3 and UNC-50 regulate the receptor expression level. RIC-3 (resistant to inhibitors of cholinesterase 3) has been shown to diversify by its differential mRNA splicing patterns. How such diversity influences neonicotinoid sensitivity of nAChRs of beneficial insect species remains poorly understood. We have identified a RIC-3 variant expressed most abundantly in the thoracic ganglia of honeybee (Apis mellifera) workers and investigated its effects on the functional expression and pharmacology of Amα1/Amα8/Amβ1 and Amα1/Amα2/Amα8/Amβ1 nAChRs expressed in X. laevis oocytes. The AmRIC-3 enhanced the response amplitude to the acetylcholine (ACh) of these A. mellifera nAChRs when its cRNAwas injected into oocytes at low concentrations but suppressed the ACh response amplitude at high concentrations. Co-expression of the AmRIC-3 had a minimal impact on the affinity of ACh, but changed the efficacy of imidacloprid and clothianidin, suggesting that the presence and the level of RIC-3 expression can affect the nAChR responses to ACh and neonicotinoids, depending on nAChR subunit composition in honeybees. © 2024 Society of Chemical Industry.

Structural switch in acetylcholine receptors in developing muscle.

During development, motor neurons originating in the brainstem and spinal cord form elaborate synapses with skeletal muscle fibres1. These neurons release acetylcholine (ACh), which binds to nicotinic ACh receptors (AChRs) on the muscle, initiating contraction. Two types of AChR are present in developing muscle cells, and their differential expression serves as a hallmark of neuromuscular synapse maturation2-4. The structural principles underlying the switch from fetal to adult muscle receptors are unknown. Here, we present high-resolution structures of both fetal and adult muscle nicotinic AChRs, isolated from bovine skeletal muscle in developmental transition. These structures, obtained in the absence and presence of ACh, provide a structural context for understanding how fetal versus adult receptor isoforms are tuned for synapse development versus the all-or-none signalling required for high-fidelity skeletal muscle contraction. We find that ACh affinity differences are driven by binding site access, channel conductance is tuned by widespread surface electrostatics and open duration changes result from intrasubunit interactions and structural flexibility. The structures further reveal pathogenic mechanisms underlying congenital myasthenic syndromes.

Origin of acetylcholine antagonism in ELIC, a bacterial pentameric ligand-gated ion channel

ELIC is a prokaryotic homopentameric ligand-gated ion channel that is homologous to vertebrate nicotinic acetylcholine receptors. Acetylcholine binds to ELIC but fails to activate it, despite bringing about conformational changes indicative of activation. Instead, acetylcholine competitively inhibits agonist-activated ELIC currents. What makes acetylcholine an agonist in an acetylcholine receptor context, and an antagonist in an ELIC context, is not known. Here we use available structures and statistical coupling analysis to identify residues in the ELIC agonist-binding site that contribute to agonism. Substitution of these ELIC residues for their acetylcholine receptor counterparts does not convert acetylcholine into an ELIC agonist, but in some cases reduces the sensitivity of ELIC to acetylcholine antagonism. Acetylcholine antagonism can be abolished by combining two substitutions that together appear to knock out acetylcholine binding. Thus, making the ELIC agonist-binding site more acetylcholine receptor-like, paradoxically reduces the apparent affinity for acetylcholine, demonstrating that residues important for agonist binding in one context can be deleterious in another. These findings reinforce the notion that although agonism originates from local interactions within the agonist-binding site, it is a global property with cryptic contributions from distant residues. Finally, our results highlight an underappreciated mechanism of antagonism, where agonists with appreciable affinity, but negligible efficacy, present as competitive antagonists.

Effects of cofactors RIC-3, TMX3 and UNC-50, together with distinct subunit ratios on the agonist actions of imidacloprid on Drosophila melanogaster Dα1/Dβ1 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dβ1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dβ1 nAChRs when expressed in X. laevis oocytes. We have investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dβ1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dβ1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dβ1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1-Dβ1 orthosteric site over the Dα1-Dα1 orthosteric site.

Stoichiometry-Selective Antagonism of α4β2 Nicotinic Acetylcholine Receptors by Fluoroquinolone Antibiotics.

Quinolone antibiotics disrupt bacterial DNA synthesis by interacting with DNA gyrase and topoisomerase IV. However, in addition, they have been shown to act as inhibitors of pentameric ligand-gated ion channels such as GABAA receptors and the α7 nicotinic acetylcholine receptor (nAChR). In the present study, we have examined the effects of quinolone antibiotics on the human α4β2 nAChR, an important subtype that is widely expressed in the central nervous system. A key feature of α4β2 nAChRs is their ability to coassemble into two distinct stoichiometries, (α4)2(β2)3 and (α4)3(β2)2, which results in differing affinities for acetylcholine. The effects of nine quinolone antibiotics were examined on both stoichiometries of the α4β2 receptor by two-electrode voltage-clamp recording. All compounds exhibited significant inhibition of α4β2 nAChRs. However, all of the fluoroquinolone antibiotics examined (ciprofloxacin, enoxacin, enrofloxacin, difloxacin, norfloxacin, pefloxacin, and sparfloxacin) were significantly more potent inhibitors of (α4)2(β2)3 nAChRs than of (α4)3(β2)2 nAChRs. This stoichiometry-selective effect was most pronounced with pefloxacin, which inhibited (α4)2(β2)3 nAChRs with an IC50 of 26.4 ± 3.4 μM but displayed no significant inhibition of (α4)3(β2)2 nAChRs. In contrast, two nonfluorinated quinolone antibiotics (cinoxacin and oxolinic acid) exhibited no selectivity in their inhibition of the two stoichiometries of α4β2. Computational docking studies suggest that pefloxacin interacts selectively with an allosteric transmembrane site at the β2(+)/β2(−) subunit interface, which is consistent with its selective inhibition of (α4)2(β2)3. These findings concerning the antagonist effects of fluoroquinolones provide further evidence that differences in the subunit stoichiometry of heteromeric nAChRs can result in substantial differences in pharmacological properties.

Investigating Drivers for M1 Muscarinic Acetylcholine Receptor‐Mediated Adverse Events by M1 Positive Allosteric Modulators

IntroductionTargeting the allosteric site of the M1 muscarinic acetylcholine receptor (M1 mAChR) is a promising strategy to develop selective drugs to treat cognitive disorders. By binding to the allosteric site, positive allosteric modulators of the M1 mAChRs (M1 PAMs) can enhance the binding (α) and/or the efficacy (β) of the endogenous ligand acetylcholine (ACh) (Fig 1), therefore improving impaired cholinergic transmission that is evident in Alzheimer’s disease and Schizophrenia. In addition, M1 PAMs may also display direct allosteric agonism (tB) (Fig 1). To date, many highly selective M1 PAMs with diverse structures have been developed, unfortunately, the majority of them still caused M1‐mediated cholinergic adverse effects (AEs), including gastrointestinal (GI) disturbances, salivation, increased heart rate and convulsions. This results from the ability of the M1 mAChR to interact with multiple intracellular partners, leading to different functional outcomes, both the beneficial (i.e, improved cognition), and the detrimental (i.e, GI AEs) effects. Excitingly, M1 PAMs can bias the M1 mAChR towards specific signalling outcomes versus others, a phenomenon called biased modulation. However, to date, it is unknown which intracellular partner is the most predominant in M1 PAM activity, and how this may drive the preclinically observed AEs.AimTo determine the role(s) Gα protein(s) and β‐arrestins exert on the allosteric activity of 5 structurally distinct M1 PAMs and link these to their reported AEs.MethodsThe M1 PAMs including BQCA, MK7622, PF06767832, MIPS1780 which showed AEs in in vivo studies, and VU0486846 without observed AEs, were assessed against ACh, in parental or CRISPR HEK293A cells with deletion of specific G proteins or β‐arrestins (ΔGαq/11, ΔGα12/13or Δβ‐arrestin 1/2) expressing the human M1 mAChR. [3H]‐NMS binding assays were performed to determine the binding affinity of ACh (KA), and of the 5 M1 PAMs (KB) and their binding cooperativity (α) with ACh. IP1 accumulation and b‐arrestin 2 recruitment were performed to quantify the efficacy (τB) of the M1 PAMs and their functional cooperativity (αβ) with ACh.ResultsΔGαq/11, ΔGα12/13 or Δb‐arrestin 1/2 significantly reduced the binding affinity of ACh, and for 3 M1‐PAMs, MK7622, PF06767832 and MIPS1780. Interestingly, all these deletions significantly increased the binding cooperativity of these M1‐PAMs with ACh. Unsurprisingly, ΔGαq/11 completely abolished IP1 responses from both orthosteric and allosteric ligands. Notably, ΔGα12/13 or Δβ‐arrestin 1/2 both increased ACh efficacy (τA). While ΔG12/13 caused no change in efficacy of tested PAMs but increased functional cooperativity of PF06767832 and MIPS1780 with ACh, Δβ‐arrestin 1/2 affected the functional properties (τB and αβ) of MK7622 in IP1 pathway. In β‐arrestin 2 pathway, ΔGαq/11 increased MIPS1780 efficacy and ΔGα12/13 increased functional cooperativity of BQCA.ConclusionGαq/11, Gα12/13 and b‐arrestin 1/2 influence binding affinity and functional properties for both ACh and M1 PAMs. These transductors affect M1 PAMs with observed AEs more than the M1 PAM without AEs, VU0486846, and thus playing a potential role in driving AEs.

Expression, purification and preliminary characterisation of the choline transporter LicB from opportunistic bacterial pathogens

Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-β-d-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family.

The pharmacologic characterization of allosteric molecules: Gq protein activation

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo.Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales.Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1–(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration–response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (β cooperativity).Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model.Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

Modeling effects of voltage dependent properties of the cardiac muscarinic receptor on human sinus node function.

The cardiac muscarinic receptor (M2R) regulates heart rate, in part, by modulating the acetylcholine (ACh) activated K+ current IK,ACh through dissociation of G-proteins, that in turn activate KACh channels. Recently, M2Rs were noted to exhibit intrinsic voltage sensitivity, i.e. their affinity for ligands varies in a voltage dependent manner. The voltage sensitivity of M2R implies that the affinity for ACh (and thus the ACh effect) varies throughout the time course of a cardiac electrical cycle. The aim of this study was to investigate the contribution of M2R voltage sensitivity to the rate and shape of the human sinus node action potentials in physiological and pathophysiological conditions. We developed a Markovian model of the IK,ACh modulation by voltage and integrated it into a computational model of human sinus node. We performed simulations with the integrated model varying ACh concentration and voltage sensitivity. Low ACh exerted a larger effect on IK,ACh at hyperpolarized versus depolarized membrane voltages. This led to a slowing of the pacemaker rate due to an attenuated slope of phase 4 depolarization with only marginal effect on action potential duration and amplitude. We also simulated the theoretical effects of genetic variants that alter the voltage sensitivity of M2R. Modest negative shifts in voltage sensitivity, predicted to increase the affinity of the receptor for ACh, slowed the rate of phase 4 depolarization and slowed heart rate, while modest positive shifts increased heart rate. These simulations support our hypothesis that altered M2R voltage sensitivity contributes to disease and provide a novel mechanistic foundation to study clinical disorders such as atrial fibrillation and inappropriate sinus tachycardia.

The voltage-sensitive cardiac M2 muscarinic receptor modulates the inward rectification of the G protein-coupled, ACh-gated K+ current.

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants.

Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117–119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.

The impact of low-frequency, low-force cyclic stretching of human bronchi on airway responsiveness.

BackgroundIn vivo, the airways are constantly subjected to oscillatory strain (due to tidal breathing during spontaneous respiration) and (in the event of mechanical ventilation) positive pressure. This exposure is especially problematic for the cartilage-free bronchial tree. The effects of cyclic stretching (other than high-force stretching) have not been extensively characterized. Hence, the objective of the present study was to investigate the functional and transcriptional response of human bronchi to repetitive mechanical stress caused by low-frequency, low-force cyclic stretching.MethodsAfter preparation and equilibration in an organ bath, human bronchial rings from 66 thoracic surgery patients were stretched in 1-min cycles of elongation and relaxation over a 60-min period. For each segment, the maximal tension corresponded to 80% of the reference contraction (the response to 3 mM acetylcholine). The impact of cyclic stretching (relative to non-stretched controls) was examined by performing functional assessments (epithelium removal and incubation with sodium channel agonists/antagonists or inhibitors of intracellular pathways), biochemical assays of the organ bath fluid (for detecting the release of pro-inflammatory cytokines), and RT-PCR assays of RNA isolated from tissue samples.ResultsThe application of low-force cyclic stretching to human bronchial rings for 60 min resulted in an immediate, significant increase in bronchial basal tone, relative to non-cyclic stretching (4.24 ± 0.16 g vs. 3.28 ± 0.12 g, respectively; p < 0.001). This cyclic stimulus also increased the affinity for acetylcholine (−log EC50: 5.67 ± 0.07 vs. 5.32 ± 0.07, respectively; p p < 0.001). Removal of airway epithelium and pretreatment with the Rho-kinase inhibitor Y27632 and inward-rectifier K+ or L-type Ca2+ channel inhibitors significantly modified the basal tone response. Exposure to L-NAME had opposing effects in all cases. Pro-inflammatory pathways were not involved in the response; cyclic stretching up-regulated the early mRNA expression of MMP9 only, and was not associated with changes in organ bath levels of pro-inflammatory mediators.ConclusionLow-frequency, low-force cyclic stretching of whole human bronchi induced a myogenic response rather than activation of the pro-inflammatory signaling pathways mediated by mechanotransduction.

Molecular Determinants of Partial agonist Affinity in Adult Neuromuscular Acetylcholine Receptors

Adult neuromuscular acetylcholine receptor-channels have two agonist binding sites located at α-δ and α-e subunit interfaces. In α, W149, Y190, and Y198 (known as the aromatic ‘triad’) are the main determinants of acetylcholine affinity (Purohit et al., 2012). Ligands resembling the components of ACh, tetramethylammonium (TMA) and choline (Cho), are partial agonists. It is unknown what interactions at the binding site distinguish full vs. partial agonists. We used single-channel electrophysiology to estimate the binding energy (resting affinity) of mutants of the triad and non-α residues eY104/δY106, eS117/δT119 and eL119/δL121 activated by partial agonists. All triad mutations weakened the affinity of the 3 agonists, in the order of αY190>αW149≈αY198. αY190F produced the same, large reduction in affinity for ACh, TMA, Cho and 3 other choline derivatives. Dissimilarities between agonists were observed with αW149F and triad Ala mutants. ACh affinity depends on the type of aromatic residue at position 149, but not Cho. The removal of the aromatic group (to Ala) had a greater effect on ACh and TMA vs. Cho. The smaller change in binding energy of Cho with αW149 mutants is consistent with the hypothesis that Cho forms an H-bond with the αW149 backbone (Bruhova et al., 2013). On the non-α subunits, eY104/δY106 mutations (A, S and L) enhanced ACh, TMA and Cho affinities, but eS117/δT119 or eL119/δL121 mutations weakly reduced affinity. The role of eY104 is explained by a molecular model in which eY104 π-stacks with αW149 to weaken the interaction of this indole with the agonist's quaternary amine. In agreement, experiments showed strong energy coupling in the double mutant eY104A-αW149A. This also explains, in part, why the fetal receptor containing a Leu at γY104 has a higher affinity for ACh and Cho. Supported by CIHR and NS-64969.

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.

Short-Term Desensitization of Muscarinic K+ Current in the Heart

Acetylcholine (ACh) rapidly increases cardiac K+ currents (IKACh) by activating muscarinic K+ (KACh) channels followed by a gradual amplitude decrease within seconds. This phenomenon is called short-term desensitization and its precise mechanism and physiological role are still unclear. We constructed a mathematical model for IKACh to examine the conditions required to reconstitute short-term desensitization. Two conditions were crucial: two distinct muscarinic receptors (m2Rs) with different affinities for ACh, which conferred an IKACh response over a wide range of ACh concentrations, and two distinct KACh channels with different affinities for the G-protein βγ subunits, which contributed to reconstitution of the temporal behavior of IKACh. Under these conditions, the model quantitatively reproduced several unique properties of short-term desensitization observed in myocytes: 1), the peak and quasi-steady states with 0.01–100 μM [ACh]; 2), effects of ACh preperfusion; and 3), recovery from short-term desensitization. In the presence of 10 μM ACh, the IKACh model conferred recurring spontaneous firing after asystole of 8.9 s and 10.7 s for the Demir and Kurata sinoatrial node models, respectively. Therefore, two different populations of KACh channels and m2Rs may participate in short-term desensitization of IKACh in native myocytes, and may be responsible for vagal escape at nodal cells.

Asymmetric transmitter binding sites of fetal muscle acetylcholine receptors shape their synaptic response

Neuromuscular acetylcholine receptors (AChRs) have two transmitter binding sites: at α-δ and either α-γ (fetal) or α-ε (adult) subunit interfaces. The γ-subunit of fetal AChRs is indispensable for the proper development of neuromuscular synapses. We estimated parameters for acetylcholine (ACh) binding and gating from single channel currents of fetal mouse AChRs expressed in tissue-cultured cells. The unliganded gating equilibrium constant is smaller and less voltage-dependent than in adult AChRs. However, the α-γ binding site has a higher affinity for ACh and provides more binding energy for gating compared with α-ε; therefore, the diliganded gating equilibrium constant at -100 mV is comparable for both receptor subtypes. The -2.2 kcal/mol extra binding energy from α-γ compared with α-δ and α-ε is accompanied by a higher resting affinity for ACh, mainly because of slower transmitter dissociation. End plate current simulations suggest that the higher affinity and increased energy from α-γ are essential for generating synaptic responses at low pulse [ACh].

Function of Interfacial Prolines at the Transmitter-binding Sites of the Neuromuscular Acetylcholine Receptor

The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (α-ε and α-δ). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (ΔG0) and the affinity change for ACh that increases the open channel probability (ΔGB). The effects of mutations of ProD2 (εPro-121/δPro-123) were greater than those of its neighbor (εPro-120/δPro-122) and were greater at α-ε versus α-δ. The main consequence of the congenital myasthenic syndrome mutation εProD2-L was to impair the establishment of a high affinity for ACh and thus make ΔGB less favorable. At both binding sites, most ProD2 mutations decreased constitutive activity (increased ΔG0). LRYHQG and RL substitutions reduced substantially the net binding energy (made ΔGB(ACh) less favorable) by ≥2 kcal/mol at α-ε and α-δ, respectively. Mutant cycle analyses were used to estimate energy coupling between the two ProD2 residues and between each ProD2 and glycine residues (αGly-147 and αGly-153) on the primary (α subunit) side of each binding pocket. The distant binding site prolines interact weakly. ProD2 interacts strongly with αGly-147 but only at α-ε and only when ACh is present. The results suggest that in the low to-high affinity change there is a concerted inter-subunit strain in the backbones at εProD2 and αGly-147. It is possible to engineer receptors having a single functional binding site by using a α-ε or α-δ ProD2-R knock-out mutation. In adult-type ACh receptors, the energy from the affinity change for ACh is approximately the same at the two binding sites (approximately -5 kcal/mol).

Defective N-linked protein glycosylation pathway in congenital myasthenic syndromes

Defective N-linked protein glycosylation pathway in congenital myasthenic syndromes

The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

BackgroundNicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit.ResultsA full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments.ConclusionData are presented demonstrating the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.

Physiological characterization of human muscle acetylcholine receptors from ALS patients

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: "microtransplantation" of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease.