Mouse Muscle Nicotinic Acetylcholine Receptor Research Articles

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 μM) than d-TC (IC50 = 0.39 μM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 μM and 7.77 μM, respectively), but BBIQA2 was similar (IC50 = 5.52 μM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1β1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75–3.08 μM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1β1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3β2, α4β2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3β2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 μM) concentration, but BBIQAs1 and 2 were less potent (only 40–50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1β3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

The atypical cation-conduction and gating properties of ELIC underscore the marked functional versatility of the pentameric ligand-gated ion-channel fold.

The superfamily of pentameric ligand-gated ion channels (pLGICs) is unique among ionotropic receptors in that the same overall structure has evolved to generate multiple members with different combinations of agonist specificities and permeant-ion charge selectivities. However, aside from these differences, pLGICs have been typically regarded as having several invariant functional properties. These include pore blockade by extracellular quaternary-ammonium cations in the micromolar-to-millimolar concentration range (in the case of the cation-selective members), and a gain-of-function phenotype, which manifests as a slower deactivation time course, as a result of mutations that reduce the hydrophobicity of the transmembrane pore lining. Here, we tested this notion on three distantly related cation-selective members of the pLGIC superfamily: the mouse muscle nicotinic acetylcholine receptor (nAChR), and the bacterial GLIC and ELIC channels. Remarkably, we found that, whereas low millimolar concentrations of TMA(+) and TEA(+) block the nAChR and GLIC, neither of these two quaternary-ammonium cations blocks ELIC at such concentrations; instead, both carry measurable inward currents when present as the only cations on the extracellular side. Also, we found that, whereas lidocaine binding speeds up the current-decay time courses of the nAChR and GLIC in the presence of saturating concentrations of agonists, the binding of lidocaine to ELIC slows this time course down. Furthermore, whereas mutations that reduce the hydrophobicity of the side chains at position 9' of the M2 α-helices greatly slowed the deactivation time course of the nAChR and GLIC, these mutations had little effect--or even sped up deactivation--when engineered in ELIC. Our data indicate that caution should be exercised when generalizing results obtained with ELIC to the rest of the pLGICs, but more intriguingly, they hint at the possibility that ELIC is a representative of a novel branch of the superfamily with markedly divergent pore properties despite a well-conserved three-dimensional architecture.

16-Morpholino quaternary ammonium steroidal derivatives as neuromuscular blocking agents: Synthesis, biological evaluation and in silico probe of ligand–receptor interaction

A series of steroidal 3,16-bis-quaternary ammonium salts were synthesized and screened on mouse hemi-diaphragm to explore new steroidal neuromuscular blocking agents. There were two compounds, 3β-piperidino derivate 8d (IC50 = 3.49 μM) and 3β-N-methylbenzylamino derivate 8g (IC50 = 4.54 μM), showing activity close to rocuronium (IC50 = 2.50 μM). The preliminary structure–activity relationship was deduced from the bioactivity results with the aid of the calculated N–N distance and log P. Meanwhile, the interactions between the ligand and binding pocket were revealed by docking 8d to the ligand binding domain of the mouse muscle nicotinic acetylcholine receptor (nAChR). This nAChR was modeled using Molecular Operating Environment (MOE) package indirectly from mollusca acetylcholine binding protein with mouse neuron α7 nAChR as intermediary template.

αC-Conotoxin PrXA: A New Family of Nicotinic Acetylcholine Receptor Antagonists

We have purified a novel paralytic peptide with 32 AA and a single disulfide bond from the venom of Conus parius, a fish-hunting species. The peptide has the following sequence: TYGIYDAKPOFSCAGLRGGCVLPONLROKFKE-NH2, where O is 4-trans-hydroxyproline. The peptide, designated alphaC-conotoxin PrXA (alphaC-PrXA), is the defining member of a new, structurally distinct family of Conus peptides. The peptide is a competitive nAChR antagonist; all previously characterized conotoxins that competitively antagonize nAChRs are structurally and genetically unrelated. (Most belong to the alpha- and alphaA-conotoxin families.) When administered to mice and fish in vivo, alphaC-PrXA caused paralysis and death. In electrophysiological assays, alphaC-PrXA potently antagonized mouse muscle nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.8 and 3.0 nM for the adult (alpha1beta1 epsilondelta subunits) and fetal (alpha1beta1 gammadelta subunits) muscle nAChR subtypes, respectively. When tested on a variety of ligand-gated and voltage-gated ion channels, alphaC-PrXA proved to be a highly specific inhibitor of the neuromuscular nAChR. The peptide competes with alpha-bungarotoxin for binding at the alpha/delta and alpha/gamma subunit interfaces of the nAChR, with higher affinity for the alpha/delta subunit interface. AlphaC-PrXA is strikingly different from the many conopeptides shown to be nicotinic antagonists; it is most similar in its general biochemical features to the snake toxins known as Waglerins.

Two Potent α3/5 Conotoxins from Piscivorous <italic>Conus achatinus</italic>

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. alpha3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of alpha3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac1.1a and Ac1.1b, with appropriate disulfide bonding, based on cDNA sequences of alpha3/5 conotoxins from C. achatinus. Ac1.1a and Ac1.1b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC50 values of 36 nM and 26 nM, respectively. For Ac1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the N-terminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the alpha1-delta subunit interface instead of the alpha1-gamma binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.

The role of the amino acid residue at α1:189 in the binding of neuromuscular blocking agents to mouse and human muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites. A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [(125)I]alpha-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant alpha1 subunits. The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC(50) results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC(50) values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at alpha1:189 and proline at alpha1:194 to serine. However the alpha1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs. The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The alpha1:Phe189Ser substitution, but not alpha1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC(50) values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary 'reinforcement'.

Pitrazepin: Interactions with Transmitter Receptors of the Central and Peripheral Nervous Systems

Pitrazepin [3-(piperazinyl-1)-9H-dibenz(c.f)triazolo(4,5-a)azepin], is an N-aryl-piperazine derivative, whose heterocyclic structure is similar to that of atypical antipsychotics like clozapine. This paper is an overview of the pharmacological actions of pitrazepin, with an emphasis on its actions on GABAA and glycine neurotransmitter receptors. Recent results on the antagonist actions of pitrazepin on acetylcholine (ACh) currents in oocytes expressing either rat neuronal or mouse muscle nicotinic acetylcholine receptors are presented. Many of the pharmacological features of the new-generation antipsychotics have been derived from studies of structurally treated drugs. A detailed understanding of the ineractions of these drugs with neurotransmitter receptor is necessary to develop more effective antipsychotics.

A Unified View of the Role of Electrostatic Interactions in Modulating the Gating of Cys Loop Receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.

Allosteric Modulation of the Nicotinic Acetylcholine Receptor by Physostigmine

The action of physostigmine on mouse muscle nicotinic acetylcholine receptor expressed in the COS-7 cell line was studied by the patch-clamp technique. Physostigmine accelerated, by allosteric modulation, the rate of desensitization of whole cell currents induced by acetylcholine and decreased the maximal amplitude in concentration-dependent manner.

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

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

Investigation of apparent mass deviations in electrospray ionization tandem mass spectrometry of a benzophenone-labeled peptide.

In a previous study utilizing benzophenone-based topological probes to study conformationally dependent changes in mouse muscle nicotinic acetylcholine receptor (nAChR) topology, electrospray ionization tandem mass spectrometric (ESI-MS/MS) analysis led to a consistent -2.0 Da mass deviation from expected values. In the present study a synthetic peptide, corresponding to nAChR alpha1 subunit residues 130-139, was photolabeled. MS/MS analysis of this peptide using an ion trap confirmed the previously observed mass deviation, associated only with fragment ions that contain the incorporated benzophenone moiety. Analysis of peak profiles for the photolabeled ions does not indicate the typical 'peak fronting' that produces a mass shift when labile ions are prematurely ejected from the ion trap. Rather, hydrogen/deuterium (H/D) exchange experiments support the hypothesis that a chemical rearrangement involving phenyl migration and ketone formation has formed an unexpected oxidized peptide, with molecular mass 2 Da less than that expected, that is isolated for collision-induced dissociation in the ion trap together with the predicted precursor due to the broad ion isolation window specified.

Characterization of the interactions between volatile anesthetics and neuromuscular blockers at the muscle nicotinic acetylcholine receptor.

Volatile anesthetics enhance the neuromuscular blockade produced by nondepolarizing muscle relaxants (NDMRs). The neuromuscular junction is a postulated site of this interaction. We tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptor. The adult mouse muscle nicotinic acetylcholine receptor (alpha(2), beta, delta, epsilon) was heterologously expressed in Xenopus laevis oocytes. Concentration-effect curves for the inhibition of acetylcholine-induced currents were established for vecuronium, d-tubocurarine, isoflurane, and sevoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the volatile anesthetics at a concentration equivalent to half the concentration producing a 50% inhibition alone. All individually tested compounds produced rapid and readily reversible concentration-dependent inhibition. The calculated 50% inhibitory concentration values were 9.9 nM (95% confidence interval [CI], 8.4-11.4 nM), 43.4 nM (95% CI, 33.6-53.3 nM), 897 microM (95% CI, 699-1150 microM), and 818 microM (95% CI, 685-1001 microM) for vecuronium, d-tubocurarine, isoflurane, and sevoflurane, respectively. Coapplication of either isoflurane or sevoflurane significantly enhanced the inhibitory effects of vecuronium and d-tubocurarine, especially so at small concentrations of NDMRs. Volatile anesthetics increase the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site. This effect may contribute to the clinically observable enhancement of neuromuscular blockade by volatile anesthetics. Isoflurane and sevoflurane enhance the receptor blocking effects of nondepolarizing muscle relaxants on nicotinic acetylcholine receptors.

Characterization of the Interactions Between Volatile Anesthetics and Neuromuscular Blockers at the Muscle Nicotinic Acetylcholine Receptor

Volatile anesthetics enhance the neuromuscular blockade produced by nondepolarizing muscle relaxants (NDMRs). The neuromuscular junction is a postulated site of this interaction. We tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptor. The adult mouse muscle nicotinic acetylcholine receptor (α2, β, δ, ε) was heterologously expressed in Xenopus laevis oocytes. Concentration-effect curves for the inhibition of acetylcholine-induced currents were established for vecuronium, d-tubocurarine, isoflurane, and sevoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the volatile anesthetics at a concentration equivalent to half the concentration producing a 50% inhibition alone. All individually tested compounds produced rapid and readily reversible concentration-dependent inhibition. The calculated 50% inhibitory concentration values were 9.9 nM (95% confidence interval [CI], 8.4–11.4 nM), 43.4 nM (95% CI, 33.6–53.3 nM), 897 μM (95% CI, 699–1150 μM), and 818 μM (95% CI, 685–1001 μM) for vecuronium, d-tubocurarine, isoflurane, and sevoflurane, respectively. Coapplication of either isoflurane or sevoflurane significantly enhanced the inhibitory effects of vecuronium and d-tubocurarine, especially so at small concentrations of NDMRs. Volatile anesthetics increase the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site. This effect may contribute to the clinically observable enhancement of neuromuscular blockade by volatile anesthetics.

Desensitization of diliganded mouse muscle nicotinic acetylcholine receptor channels.

Nicotinic ACh receptor channels (AChRs) exposed to high concentrations of ACh adopt 'desensitized' conformations that have a high affinity for the transmitter and no measurable ion conductance. Single-channel currents elicited by 0.1 or 1 mM ACh were recorded from human embryonic kidney (HEK) cells that had been transiently transfected with mouse alpha, beta, delta, and epsilon subunits. On the time scale of approximately 0.1 ms to approximately 1 h, apparent open intervals are described by a single exponential component, and shut intervals associated with desensitization are described by the sum of four or five exponential components. The kinetic behaviour appeared to be stationary and homogeneous. Desensitization rate constants were estimated by kinetic modelling of currents from cell-attached and outside-out patches (where the number of channels in the patch was measured). A single AChR recovered from the longest-lived desensitized state only after approximately 5 min. The occupancy of an AChR for each of the desensitized states was calculated as a function of time after the continuous application of a pulse of saturating ACh. The longest-lived desensitized state accounted for 90 % of the total only after several seconds. The fractional recovery from desensitization (during a 200 ms wash period) decreased as the duration of the desensitizing pulse increased, suggesting that recovery is slower from the longer-lived desensitized states. The free energy landscape for the AChR desensitization reaction in cell-attached patches exhibited an initial destabilization, followed by a plateau region of gradually increasing stability, followed by a deep well.

Isobolographic analysis of non-depolarising muscle relaxant interactions at their receptor site

Administration of certain combinations of non-depolarising muscle relaxants produces greater than expected neuromuscular blockade. Synergistic effects may be explained by drug interactions with the postsynaptic muscle nicotinic acetylcholine receptor. To investigate this hypothesis, the adult mouse muscle nicotinic acetylcholine receptor (α 2βδε) was heterologously expressed in Xenopus laevis oocytes and activated by the application of acetylcholine (10 μM). The effects of five individually applied muscle relaxants and six combinations of structurally similar and dissimilar compounds were studied. Drug combinations containing equipotent concentrations of two agents were tested and dose–response curves were determined. All compounds tested alone and in combination produced rapid and readily reversible, concentration-dependent inhibition. Isobolographic and fractional analyses indicated additive interactions for all six tested combinations. These findings suggest that synergistic neuromuscular blocking effects, observed for the administration of certain combinations of muscle relaxants, do not result from purely postsynaptic binding events at the muscle nicotinic acetylcholine receptor, but rather from differential actions on pre- and postsynaptic sites.

Antagonism of nicotinic acetylcholine receptors by inhibitors of monoamine uptake.

A study was made of the effects of several monoamine-uptake inhibitors on membrane currents elicited by acetylcholine (ACh-currents) generated by rat neuronal alpha2beta4 and mouse muscle nicotinic acetylcholine receptors (AChRs) expressed in Xenopus laevis oocytes. For the two types of receptors the monoamine-uptake inhibitors reduced the ACh-currents albeit to different degrees. The order of inhibitory potency was norfluoxetine > clomipramine > indatraline > fluoxetine > imipramine > zimelidine > 6-nitro-quipazine > trazodone for neuronal alpha2beta4 AChRs, and norfluoxetine > fluoxetine > imipramine > clomipramine > indatraline > zimelidine > trazodone > 6-nitro-quipazine for muscle AChRs. Thus, the most potent inhibitor was norfluoxetine, whilst the weakest ones were trazodone, 6-nitro-quipazine and zimelidine. Effects of the tricyclic antidepressant imipramine were studied in more detail. Imipramine inhibited reversibly and non-competitively the ACh-current with a similar inhibiting potency for both neuronal alpha2beta4 and muscle AChRs. The half-inhibitory concentrations of imipramine were 3.65 +/- 0.30 microM for neuronal alpha2beta4 and 5.57 +/- 0.19 microM for muscle receptors. The corresponding Hill coefficients were 0.73 and 1.2 respectively. The inhibition of imipramine was slightly voltage-dependent, with electric distances of approximately 0.10 and approximately 0.12 for neuronal alpha2beta4 and muscle AChRs respectively. Moreover, imipramine accelerated the rate of decay of ACh- currents of both muscle and neuronal AChRs. The ACh-current inhibition was stronger when oocytes, expressing neuronal alpha2beta4 or muscle receptors, were preincubated with imipramine alone than when it was applied after the ACh-current had been generated, suggesting that imipramine acts also on non-activated or closed AChRs. We conclude that monoamine-uptake inhibitors reduce ACh-currents and that imipramine regulates reversibly and non- competitively neuronal alpha2beta4 and muscle AChRs through similar mechanisms, perhaps by interacting externally on a non-conducting state of the AChR and by blocking the open receptor-channel complex close to the vestibule of the channel. These studies may be important for understanding the regulation of AChRs as well as for understanding antidepressant- and side-effects of monoamine-uptake inhibitors.

Zinc permeates mouse muscle ACh receptor channels expressed in BOSC 23 cells and affects channel function

1. The influx of Zn2+ through the channels of fetal and adult mouse muscle nicotinic acetylcholine receptors (gamma- and epsilon-AChRs) and its effects on receptor function were studied in transiently transfected human BOSC 23 cells, by combining patch-clamp recordings with digital fluorescence microscopy. 2. ACh-induced whole-cell currents were reversibly reduced by external ZnCl2, with half-maximal inhibitory concentrations of 3 and 1 mM for gamma- and epsilon-AChRs, respectively. 3. Both gamma- and epsilon-AChR channels were permeable to Zn2+, as shown by fluorescence measurements using Zn2+-sensitive dyes. The fractional current carried by Zn2+ (Pf,Zn; 0.5 mM Zn2+ in Ca2+- and Mg2+-free medium) through gamma- and epsilon">-AChR channels was 1.7 and 4 %, respectively. 4. Pf,Zn increased with the concentration of ZnCl2, but was little affected by physiological concentrations of Ca2+ and Mg2+ in the external medium. 5. The conductance of ACh-evoked unitary events, measured by cell-attached or outside-out recordings, decreased when the patched membrane was exposed to ZnCl2 (1 or 3 mM). Simultaneous application of ACh and Zn2+ to the extra-patch membrane lengthened channel open duration (tau op) by 50%. No obvious increment of tau op was observed following exposure of inside-out patches to Zn2+. 6. The possible physiological relevance of zinc-induced modulation of AChR channels is discussed.

Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site.

We have constructed a series of cysteine-substitution mutants in order to identify residues in the mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in alpha-bungarotoxin (alpha-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that alpha subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the alpha-Bgtx binding site in native receptors. These results with the intact receptor are consistent with NMR studies of an alpha-Bgtx/receptor-dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993) Biochemistry 32, 12290-12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially between AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C with either bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylammonium-containing thiol-modifying reagents with agonist properties, results in a marked decrease ( approximately 55-70%) in the number of alpha-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both alpha/gamma and alpha/delta sites to the same extent, as shown for alphaW187C and alphaF189C which were the two mutants examined on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as methylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl methane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthiosulfonate (MTSEA), has little or no effect on the maximal binding of alpha-Bgtx to the alphaW187C, alphaV188C, or alphaF189C mutant receptors. The striking alkylammonium dependency suggests that an interaction of the tethered modifying group with the negative subsite within the agonist binding domain is primarily responsible for the observed blockade of toxin binding.

Activation and blockade of mouse muscle nicotinic channels by antibodies directed against the binding site of the acetylcholine receptor.

1. Using the patch-clamp technique, we have found that mouse muscle nicotinic acetylcholine receptor (nAChR) channels can be activated by low concentrations of a monoclonal antibody (MoAb), referred to as WF6, which is directed against the acetylcholine (ACh) binding site. Similar effects were seen using IgG or F(ab)2 fragments from the sera of patients with myasthenia gravis (MG), which contain polyclonal anti-nAChR antibodies. 2. The mean open times of MoAb and the slope conductance of single WF6-activated single channels were similar to those of ACh-activated channels under the same experimental conditions. 3. On outside-out patches, single channel activity was elicited by MoAb WF6 and MG F(ab)2 fragments, and was blocked by (+)-tubocurarine. We therefore concluded that MoAb WF6 and the MG F(ab)2 fragments activate the nAChR. 4. MoAb WF6 and MG F(ab)2 fragments blocked the current activated by pulsed application of 10(-4) M ACh to a significant extent. The block was partly reversible. The rate constants for the binding and dissociation of MoAb WF6 from the receptor were determined quantitatively.

Transgenic engineering of neuromuscular junctions in Xenopus laevis embryos transiently overexpressing key cholinergic proteins.

To examine the role of key cholinergic proteins in the formation of neuromuscular junctions (NMJs), we expressed DNAs encoding the mouse muscle nicotinic acetylcholine receptor (nAChR) or human brain and muscle acetylcholinesterase (hAChE) in developing Xenopus laevis embryos. Acetylthiocholine hydrolysis and alpha-bungarotoxin binding in homogenates of transgenic embryos revealed transient overexpression of the respective proteins for at least 4 days postfertilization. Moreover, hAChE injection induced an approximately 2-fold increase in endogenous Xenopus nAChR. Electron microscopy coupled with cytochemical staining for AChE activity revealed that AChE-stained areas, which reached 0.17 microns2 in NMJs of control embryos raised at 21 degrees C, increased up to 0.53 and 0.60 microns2 in nAChR and hAChE transgenics, respectively. These increases coincided with the appearance of a class of large NMJs with average postsynaptic lengths up to 1.8-fold greater than controls. As much as 57% and 34% of the NMJs in animals transgenic for nAChR and hAChE, respectively, displayed AChE activity in nerve terminals in addition to muscle labeling, as compared with 10% nerve-labeled NMJs in control animals. Moreover, area, but not length values, were > 2-fold larger in hAChE-expressing NMJs labeled in their nerve terminals than in those labeled in muscle alone, reflecting a hAChE-induced increase in synaptic cleft width. These findings indicate that modulation of cholinergic neurotransmission in NMJs modifies the features of nerve-muscle connections.