Muscle-type Nicotinic Receptors Research Articles

Structural interplay of anesthetics and paralytics on muscle nicotinic receptors

General anesthetics and neuromuscular blockers are used together during surgery to stabilize patients in an unconscious state. Anesthetics act mainly by potentiating inhibitory ion channels and inhibiting excitatory ion channels, with the net effect of dampening nervous system excitability. Neuromuscular blockers act by antagonizing nicotinic acetylcholine receptors at the motor endplate; these excitatory ligand-gated ion channels are also inhibited by general anesthetics. The mechanisms by which anesthetics and neuromuscular blockers inhibit nicotinic receptors are poorly understood but underlie safe and effective surgeries. Here we took a direct structural approach to define how a commonly used anesthetic and two neuromuscular blockers act on a muscle-type nicotinic receptor. We discover that the intravenous anesthetic etomidate binds at an intrasubunit site in the transmembrane domain and stabilizes a non-conducting, desensitized-like state of the channel. The depolarizing neuromuscular blocker succinylcholine also stabilizes a desensitized channel but does so through binding to the classical neurotransmitter site. Rocuronium binds in this same neurotransmitter site but locks the receptor in a resting, non-conducting state. Together, this study reveals a structural mechanism for how general anesthetics work on excitatory nicotinic receptors and further rationalizes clinical observations in how general anesthetics and neuromuscular blockers interact.

Muscle-type nicotinic acetylcholine receptor structure, function, and pharmacology.

Nicotinic acetylcholine receptors mediate fast chemical transmission throughout the central and peripheral nervous systems. The nicotinic receptor subtype found on the motor endplate at the neuromuscular junction has been an important model system for understanding receptor structure, function, signaling, and pharmacology. The nicotinic receptor found in the electric organs of fish, including the Torpedo ray, is homologous to the human muscle-type nicotinic receptor and serves as an abundant natural source for biochemical and structural studies. Here we leverage the Torpedo nicotinic receptor in structural biology and electrophysiological studies to understand how clinically relevant drug classes regulate receptor conformation and activity. The findings reveal how different types of functional antagonists stabilize distinct non-conducting states to inhibit neuromuscular transmission.

Nicotinic acetylcholine receptor structural pharmacology and gating transitions.

Our lab focuses on using structural and functional approaches to understand signaling by ligand-gated ion channels in the nervous system. A major emphasis is on understanding nicotinic receptor pharmacology from a structural perspective. Here I will present some of our recent work using the muscle-type nicotinic receptor from the Torpedo ray as a structurally tractable reference for the human receptor at the neuromuscular junction. I will focus on how toxins from animals and plants, as well as drugs used in the clinic, act on the receptor, and how we can use this information to understand state transitions that underlie ion channel gating. These studies have revealed a new class of allosteric site at the junction of the extracellular and transmembrane domains that small molecules can leverage to modulate channel desensitization. Our findings suggest that stabilizing a desensitized-like non-conducting state, through this allosteric site, is an important alternative mechanism for antagonizing the receptor.

Structural mechanism of muscle nicotinic receptor desensitization and block by curare.

Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.

Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors.

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.

Structure of the Native Muscle-type Nicotinic Receptor and Inhibition by Snake Venom Toxins

The nicotinic acetylcholine receptor, a pentameric ligand-gated ion channel, converts the free energy of binding of the neurotransmitter acetylcholine into opening of its central pore. Here we present the first high-resolution structure of the receptor type found in muscle-endplate membrane and in the muscle-derived electric tissues of fish. The native receptor was purified from Torpedo electric tissue and functionally reconstituted in lipids optimal for cryo-electron microscopy. The receptor was stabilized in a closed state by the binding of α-bungarotoxin. The structure reveals the binding of a toxin molecule ateach of two subunit interfaces in a manner that would block the binding of acetylcholine. It also reveals a closed gate in the ion-conducting pore, formed by hydrophobic amino acid side chains, located ∼60Å from the toxin binding sites. The structure provides a framework for understanding gating in ligand-gated channels and how mutations in the acetylcholine receptor cause congenital myasthenic syndromes.

Assessment of false transmitters as treatments for nerve agent poisoning

Nerve agents inhibit acetylcholinesterase (AChE), leading to a build-up of acetylcholine (ACh) and overstimulation at cholinergic synapses. Current post-exposure nerve agent treatment includes atropine to treat overstimulation at muscarinic synapses, a benzodiazepine anti-convulsant, and an oxime to restore the function of AChE. Aside from the oxime, the components do not act directly to reduce the overstimulation at nicotinic synapses. The false transmitters acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) are analogs of ACh, synthesised similarly at synapses. AMECh and ADECh are partial agonists, with reduced activity compared to ACh, so it was hypothesised the false transmitters could reduce overstimulation.Synthetic routes to AMECh and ADECh, and their precursors, monoethylcholine (MECh) and diethylcholine (DECh), were devised, allowing them to be produced easily on a laboratory-scale. The mechanism of action of the false transmitters was investigated in vitro. AMECh acted as a partial agonist at human muscarinic (M1 and M3) and muscle-type nicotinic receptors, and ADECh was a partial agonist only at certain muscarinic subtypes. Their precursors acted as antagonists at muscle-type nicotinic, but not muscarinic receptors.Administration of MECh and DECh improved neuromuscular function in the soman-exposed guinea-pig hemi-diaphragm preparation. False transmitters may therefore help reduce nerve agent induced overstimulation at cholinergic synapses.

Mechanisms of Blockade of the Muscle-Type Nicotinic Receptor by Benzocaine, a Permanently Uncharged Local Anesthetic

Most local anesthetics (LAs) are amine compounds bearing one or several phenolic rings. Many of them are protonated at physiological pH, but benzocaine (Bzc) is permanently uncharged, which is relevant because the effects of LAs on nicotinic acetylcholine (ACh) receptors (nAChRs) depend on their presence as uncharged or protonated species. The aims of this study were to assess the effects of Bzc on nAChRs and to correlate them with its binding to putative interacting sites on this receptor. nAChRs from Torpedo electroplaques were microtransplanted to Xenopus oocytes and currents elicited by ACh (IAChs), either alone or together with Bzc, were recorded at different potentials. Co-application of ACh with increasing concentrations of Bzc showed that Bzc reversibly blocked nAChRs. IACh inhibition by Bzc was voltage-independent, but the IACh rebound elicited when rinsing Bzc suggests an open-channel blockade. Besides, ACh and Bzc co-application enhanced nAChR desensitization. When Bzc was just pre-applied it also inhibited IACh, by blocking closed (resting) nAChRs. This blockade slowed down the kinetics of both the IACh activation and the recovery from blockade. The electrophysiological results indicate that Bzc effects on nAChRs are similar to those of 2,6-dimethylaniline, an analogue of the hydrophobic moiety of lidocaine. Furthermore, docking assays on models of the nAChR revealed that Bzc and DMA binding sites on nAChRs overlap fairly well. These results demonstrate that Bzc inhibits nAChRs by multiple mechanisms and contribute to better understanding both the modulation of nAChRs and how LAs elicit some of their clinical side effects.This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Aptamer Efficacies for In Vitro and In Vivo Modulation of αC-Conotoxin PrXA Pharmacology.

The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.

Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine.

To identify the molecular determinants responsible for lidocaine blockade of muscle-type nAChRs, we have studied the effects on this receptor of 2,6-dimethylaniline (DMA), which resembles lidocaine’s hydrophobic moiety. Torpedo marmorata nAChRs were microtransplanted to Xenopus oocytes and currents elicited by ACh (IACh), either alone or co-applied with DMA, were recorded. DMA reversibly blocked IACh and, similarly to lidocaine, exerted a closed-channel blockade, as evidenced by the enhancement of IACh blockade when DMA was pre-applied before its co-application with ACh, and hastened IACh decay. However, there were marked differences among its mechanisms of nAChR inhibition and those mediated by either the entire lidocaine molecule or diethylamine (DEA), a small amine resembling lidocaine’s hydrophilic moiety. Thereby, the IC50 for DMA, estimated from the dose-inhibition curve, was in the millimolar range, which is one order of magnitude higher than that for either DEA or lidocaine. Besides, nAChR blockade by DMA was voltage-independent in contrast to the increase of IACh inhibition at negative potentials caused by the more polar lidocaine or DEA molecules. Accordingly, virtual docking assays of DMA on nAChRs showed that this molecule binds predominantly at intersubunit crevices of the transmembrane-spanning domain, but also at the extracellular domain. Furthermore, DMA interacted with residues inside the channel pore, although only in the open-channel conformation. Interestingly, co-application of ACh with DEA and DMA, at their IC50s, had additive inhibitory effects on IACh and the extent of blockade was similar to that predicted by the allotopic model of interaction, suggesting that DEA and DMA bind to nAChRs at different loci. These results indicate that DMA mainly mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine, which is emulated by the hydrophilic DEA. Furthermore, it is pointed out that the hydrophobic (DMA) and hydrophilic (DEA) moieties of the lidocaine molecule act differently on nAChRs and that their separate actions taken together account for most of the inhibitory effects of the whole lidocaine molecule on nAChRs.

Multi-target-directed therapeutic potential of 7-methoxytacrine-adamantylamine heterodimers in the Alzheimer's disease treatment

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and currently there is no efficient treatment. The classic drug-design strategy based on the “one-molecule-one-target” paradigm was found to be ineffective in the case of multifactorial diseases like AD. A novel multi-target-directed ligand strategy based on the assumption that a single compound consisting of two or more distinct pharmacophores is able to hit multiple targets has been proposed as promising. Herein, we investigated 7-methoxytacrine - memantine heterodimers developed with respect to the multi-target-directed ligand theory. The spectroscopic, microscopic and cell culture methods were used for systematic investigation of the interference of the heterodimers with β-secretase (BACE1) activity, Aβ peptide amyloid fibrillization (amyloid theory) and interaction with M1 subtype of muscarinic (mAChRs), nicotinic (nAChRs) acetylcholine receptors (cholinergic theory) and N-methyl-d-aspartate receptors (NMDA) (glutamatergic theory). The drug-like properties of selected compounds have been evaluated from the point of view of blood-brain barrier penetration and cell proliferation. We have confirmed the multipotent effect of novel series of compounds. They inhibited effectively Aβ peptide amyloid fibrillization and affected the BACE1 activity. Moreover, they have AChE inhibitory potency but they could not potentiate cholinergic transmission via direct interaction with cholinergic receptors. All compounds were reported to act as an antagonist of both M1 muscarinic and muscle-type nicotinic receptors. We have found that 7-methoxytacrine - memantine heterodimers are able to hit multiple targets associated with Alzheimer's disease and thus, have a potential clinical impact for slowing or blocking the neurodegenerative process related to this disease.

Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine.

Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (IACh) in a dose-dependent manner (IC50 close to 70 μM), but unlike lidocaine, DEA did not affect IACh desensitization. IACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced IACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.

Role of the Transmembrane α-Helix M4 in the Potentiation of Pentameric Ligand-Gated Ion Channels

The gating of pentameric ligand gated ion channels (pLGICs) is sensitive to a variety of allosteric modulators that act on the transmembrane domain, including lipids. Here, we use two prokaryotic homologs, GLIC and ELIC, to examine the role of the lipid-exposed transmembrane a-helix, M4, in the allosteric regulation of pLGICs. Aromatic interactions at the interface between M4 and the adjacent a-helices, M1 and M3, drive M4 binding during folding (Haeger et al. (2010) Nat Struct Mol Biol 17:90-98). Alanine substitutions of the aromatics at this interface in GLIC weaken M4 binding and inhibit channel function, while aromatic substitutions of aliphatic residues at the same interface in ELIC promote M4 binding and potentiate channel function. The strength of M4 binding to M1/M3 governs the susceptibility of both pLGICs to the potentiating effects of a congenital myasthenic syndrome mutation, which occurs in muscle-type nicotinic receptors on the lipid-facing surface of M4 and alters lipid-protein interactions to enhance function, but has little effect on the inhibitory activity of the drug, propofol. We thus identify both M4-dependent and M4-independent pathways for transmembrane domain allosteric regulation. Our data suggest that the chemistry at the interface between M4 and M1/M3 influences the intrinsic M4 binding affinity for M1/M3 and may thus govern the susceptibility of a pLGIC to the potentiating effects of lipids.

Energetic Changes in the β Subunit during Gating of the Muscle Nicotinic Receptor

The transmitter-binding sites in the adult muscle-type nicotinic receptor are located at the interfaces between the α subunit and the δ or ε subunits, while the β subunit does not contribute to binding. The β subunit is termed a "structural" subunit as a result. We made mutations to 27 residues in the mouse muscle β subunit (2 to 4 mutations at each location) to measure the consequences on the channel opening and closing rates. We analyzed the results in terms of the effect on the ratio of the opening to closing rates (θ) and, when sufficiently large changes occurred, on the slope of the logarithmic relation between the opening rate and θ (φ). φ can be interpreted in terms of the timing during channel activation when a given residue makes an energetic contribution to gating. We examined 12 positions in the extracellular domain (ECD) of the β subunit, chosen because mutations to homologous residues in other subunits had large effects on θ. In contrast, mutations at the locations in β produced small changes in gating. We examined 15 positions in the second transmembrane (TM2) and TM2-TM3 linker regions. The majority of locations showed larger changes in θ(typically > 1 kcal/mole). Estimates of φ were consistent with the idea that the energetic contributions from residues in the β subunit occurred later than for homologous residues in the α subunit but before residues in δ. Overall, the data suggest that the ECD and the interface between the ECD and TM domains of β make little contribution to the energetics of gating. However, channel-lining residues do contribute, although the data suggest that the timing is later than for the α subunit. (NS22356 & NS72770)

Synthetic polyamines: an overview of their multiple biological activities

The binding of polyamines to a variety of receptors and other defined recognition sites has been widely reported. It is well-known that polyamines interact with aspartate, glutamate, and aromatic residues of a given receptor and/or enzyme mainly through the formation of ion bonds, since at physiological pH, protonation of amino groups is nearly complete. From this, the hypothesis arises that a polyamine may be a universal template able to recognize different receptor systems. This hypothesis suggests that both affinity and selectivity may be fine-tuned by inserting appropriate substituents onto the amine functions and by varying the methylene chain lengths between them on the polyamine backbone. In this paper, we detail several application of this design strategy aimed at discovering potent and selective polyamines able to bind neurotransmitter receptors and enzymes, such as muscarinic receptor subtypes, muscle-type nicotinic receptors and acethylcholinesterase.

Mammalian nicotinic acetylcholine receptors: from structure to function.

The classical studies of nicotine by Langley at the turn of the 20th century introduced the concept of a "receptive substance," from which the idea of a "receptor" came to light. Subsequent studies aided by the Torpedo electric organ, a rich source of muscle-type nicotinic receptors (nAChRs), and the discovery of alpha-bungarotoxin, a snake toxin that binds pseudo-irreversibly to the muscle nAChR, resulted in the muscle nAChR being the best characterized ligand-gated ion channel hitherto. With the advancement of functional and genetic studies in the late 1980s, the existence of nAChRs in the mammalian brain was confirmed and the realization that the numerous nAChR subtypes contribute to the psychoactive properties of nicotine and other drugs of abuse and to the neuropathology of various diseases, including Alzheimer's, Parkinson's, and schizophrenia, has since emerged. This review provides a comprehensive overview of these findings and the more recent revelations of the impact that the rich diversity in function and expression of this receptor family has on neuronal and nonneuronal cells throughout the body. Despite these numerous developments, our understanding of the contributions of specific neuronal nAChR subtypes to the many facets of physiology throughout the body remains in its infancy.

Activation and Block of the Adult Muscle-Type Nicotinic Receptor by Physostigmine: Single-Channel Studies

The plant-derived acetylcholinesterase inhibitor physostigmine has previously been shown to act on the nicotinic acetylcholine receptor (nAChR) causing either direct activation or potentiation of currents elicited by low concentrations of nicotinic agonists, or, at higher concentrations, channel block. We examined mouse adult-type muscle nAChR activation by physostigmine and found that channel activation by physostigmine exhibits many characteristics common with channel activity elicited by nicotinic agonists. Single-channel conductance was indistinguishable, and mutants known to slow channel closing in the presence of nicotinic agonists had a similar effect in the presence of physostigmine. However, physostigmine is a very inefficacious agonist. The presence of physostigmine did not alter the effective opening rate for a subsaturating dosage of carbachol, suggesting that physostigmine does not interact with the nicotinic agonist binding site. Mutations to a residue (alphaLys125) previously identified as part of the putative binding site for physostigmine reduced the duration of openings elicited by physostigmine, but the effects were generally small and, in most cases, nonsignificant. At higher concentrations, physostigmine blocked channel activity. Block manifested as a reduction in the mean open time and the emergence of a closed state, with a mean duration of 3 to 7 ms. The properties of block were consistent with two equivalent blocking sites per receptor with microscopic binding and unbinding rate constants for physostigmine of 20 microM(-1) s(-1) and 450 s(-1) (K(D) = 23 microM). These observations indicate that physostigmine is able to activate muscle nAChR by interacting with a site other than the nicotinic ligand binding site.

Interactions between α-conotoxin MI and the Torpedo marmorata receptor α–δ interface

The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the α–γ and α–δ subunit interfaces; α-conotoxins can bind them selectively. Moreover, we previously reported that α-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791–795]. Herein, to identify T. marmorata receptor regions involved in α-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. α-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the α/δ interface. A proposal for receptor–toxin interaction is discussed based on experimental results and docking studies.

The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues alphaS287, alphaC412, betaY441, gammaM299, gammaS460, deltaM293, deltaS297 and deltaN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [(125)I]-alpha-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC(50)), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC(50). Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.

Galantamine Activates Muscle-Type Nicotinic Acetylcholine Receptors without Binding to the Acetylcholine-Binding Site

Galantamine (Reminyl; Janssen Pharmaceutica, Titusville, NJ) belongs to a class of acetylcholinesterase inhibitors approved for symptomatic treatment of Alzheimer's disease. The drug presumably acts by raising and prolonging the profile of acetylcholine (ACh) via an inhibitory effect on the esterase. However, there is also evidence demonstrating that galantamine can activate the nicotinic ACh receptor or modulate its activation by ACh. In this study, we have examined the ability of galantamine to directly activate the muscle-type nicotinic ACh receptor or to modulate receptor activation by selected nicotinic agonists. Studies of direct activation by galantamine demonstrated that this ligand is a low-efficacy agonist of the muscle-type ACh receptor. Point mutations in the M2-M3 linker (alphaS269I) and the M2 transmembrane domain (epsilonT264P) had similar effects on receptor activation by galantamine and nicotinic agonists, suggesting that the general features of receptor activation by galantamine are similar to that in the presence of ACh. Experiments performed in the simultaneous presence of galantamine and various nicotinic ligands showed that channel activation by the nicotinic ligands studied (ACh, carbachol, and choline) was not affected by the presence of galantamine at concentrations up to 100 microm. In addition, galantamine did not reduce the initial rate of binding for 125I-alpha-bungarotoxin. These results demonstrate that galantamine does not interfere with the occupation of the nicotinic agonist binding site by ACh, carbachol, or choline. We conclude that galantamine activates the muscle-type ACh receptor by interacting with a binding site that is distinct from the site for nicotinic agonists.