Comparison of intranasal naloxone and intranasal nalmefene in a translational model assessing the impact of synthetic opioid overdose on respiratory depression and cardiac arrest.

Introduction: Using a validated translational model that quantitatively predicts opioid-induced respiratory depression and cardiac arrest, we compared cardiac arrest events caused by synthetic opioids (fentanyl, carfentanil) following rescue by intranasal (IN) administration of the µ-opioid receptor antagonists naloxone and nalmefene. Methods: This translational model was originally developed by Mann et al. (Clin Pharmacol Ther 2022) to evaluate the effectiveness of intramuscular (IM) naloxone. We initially implemented this model using published codes, reproducing the effects reported by Mann et al. on the incidence of cardiac arrest events following intravenous doses of fentanyl and carfentanil as well as the reduction in cardiac arrest events following a standard 2 mg IM dose of naloxone. We then expanded the model in terms of pharmacokinetic and µ-opioid receptor binding parameters to simulate effects of 4 mg naloxone hydrochloride IN and 3 mg nalmefene hydrochloride IN, both FDA-approved for the treatment of opioid overdose. Model simulations were conducted to quantify the percentage of cardiac arrest in 2000 virtual patients in both the presence and absence of IN antagonist treatment. Results: Following simulated overdoses with both fentanyl and carfentanil in chronic opioid users, IN nalmefene produced a substantially greater reduction in the incidence of cardiac arrest compared to IN naloxone. For example, following a dose of fentanyl (1.63 mg) producing cardiac arrest in 52.1% (95% confidence interval, 47.3-56.8) of simulated patients, IN nalmefene reduced this rate to 2.2% (1.0-3.8) compared to 19.2% (15.5-23.3) for IN naloxone. Nalmefene also produced large and clinically meaningful reductions in the incidence of cardiac arrests in opioid naïve subjects. Across dosing scenarios, simultaneous administration of four doses of IN naloxone were needed to reduce the percentage of cardiac arrest events to levels that approached those produced by a single dose of IN nalmefene. Conclusion: Simulations using this validated translational model of opioid overdose demonstrate that a single dose of IN nalmefene produces clinically meaningful reductions in the incidence of cardiac arrest compared to IN naloxone following a synthetic opioid overdose. These findings are especially impactful in an era when >90% of all opioid overdose deaths are linked to synthetic opioids such as fentanyl.

Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability ("modes"). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the ß5-ß5' linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.

Function of the M1 π-helix in endplate receptor activation and desensitization.

KEY POINTS: A conserved proline in M1 causes a kink between α and π helical segments. The kink is under greater tension in the resting versus active conformation. The kink and the agonist do not interact directly. The π-helix separates the gating functions of the extracellular and transmembrane domains. Mutations of the conserved proline and propofol increase desensitization. ABSTRACT: Nicotinic acetylcholine receptors (AChRs) switch on/off to generate transient membrane currents (C↔O; closed-open 'gating') and enter/recover from long-lived, refractory states (O↔D; 'desensitization'). The M1 transmembrane helix of the muscle endplate AChR is linked to a ß-strand of the extracellular domain that extends to a neurotransmitter binding site. We used electrophysiology to measure the effects of mutations of amino acids that are located at a proline kink in M1 that separates π and α helices, in both α (N217, V218 and P221) and non-α subunits. In related receptors, the kink is straighter and more stable in O vs. C structures (gating is 'spring-loaded'). None of the AChR kink mutations had a measureable effect on agonist affinity but many influenced the allosteric gating constant substantially. Side chains in the M1 α-helix experience extraordinarily large energy differences between C and O structures, probably because of a ∼2 Å displacement and tilt of M2 relative to M1. There is a discrete break in the character of the gating transition state between αN217 and αV218, indicating that the π-helix is a border between extracellular- and transmembrane-domain function. Mutations of the conserved M1 proline, and the anaesthetic propofol, increase a rate constant for desensitization. The results suggest that straightening of the M1 proline kink triggers AChR desensitization.

Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.

Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process.

Functional anatomy of an allosteric protein.

Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits--the transmitter-binding sites and αM2-αM3 linkers in the membrane domain--have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

Loop C and the mechanism of acetylcholine receptor-channel gating.

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation ("priming"), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site-gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.

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).

Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153.

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (K(d)) and the net binding energy from the agonist for gating (ΔG(B)) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in K(d) and essentially no change in ΔG(B). The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in K(d) but no change in ΔG(B). The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.

Sources of energy for gating by neurotransmitters in acetylcholine receptor channels.

Nicotinic acetylcholine receptors (AChRs) mediate signaling in the central and peripheral nervous systems. The AChR gating conformational change is powered by a low- to high-affinity change for neurotransmitters at two transmitter binding sites. We estimated (from single-channel currents) the components of energy for gating arising from binding site aromatic residues in the α-subunit. All mutations reduced the energy (TyrC1>>TrpB≈TyrC2>TyrA), with TyrC1 providing ~40% of the total. Considered one at a time, the fractional energy contributions from the aromatic rings were TrpB ~35%, TyrC1 ~28%, TyrC2 ~28%, and TyrA ~10%. Together, TrpB, TyrC1, and TyrC2 comprise an "aromatic triad" that provides much of the total energy from the transmitter for gating. Analysis of mutant pairs suggests that the energy contributions from some residues are nearly independent. Mutations of TyrC1 cause particularly large energy reductions because they remove two favorable and approximately equal interactions between the aromatic ring and the quaternary amine of the agonist and between the hydroxyl and αLysß7.

The intrinsic energy of the gating isomerization of a neuromuscular acetylcholine receptor channel.

Nicotinic acetylcholine receptor (AChR) channels at neuromuscular synapses rarely open in the absence of agonists, but many different mutations increase the unliganded gating equilibrium constant (E0) to generate AChRs that are active constitutively. We measured E0 for two different sets of mutant combinations and by extrapolation estimated E0 for wild-type AChRs. The estimates were 7.6 and 7.8×10(-7) in adult-type mouse AChRs (-100 mV at 23°C). The values are in excellent agreement with one obtained previously by using a completely different method (6.5×10(-7), from monoliganded gating). E0 decreases with depolarization to the same extent as does the diliganded gating equilibrium constant, e-fold with ∼60 mV. We estimate that at -100 mV the intrinsic energy of the unliganded gating isomerization is +8.4 kcal/mol (35 kJ/mol), and that in the absence of a membrane potential, the intrinsic chemical energy of this global conformational change is +9.4 kcal/mol (39 kJ/mol). Na+ and K+ in the extracellular solution have no measureable effect on E0, which suggests that unliganded gating occurs with only water occupying the transmitter binding sites. The results are discussed with regard to the energy changes in receptor activation and the competitive antagonism of ions in agonist binding.

Design and control of acetylcholine receptor conformational change.

Allosteric proteins use energy derived from ligand binding to promote a global change in conformation. The "gating" equilibrium constant of acetylcholine receptor-channels (AChRs) is influenced by ligands, mutations, and membrane voltage. We engineered AChRs to have specific values of this constant by combining these perturbations, and then calculated the corresponding values for a reference condition. AChRs were designed to have specific rate and equilibrium constants simply by adding multiple, energetically independent mutations with known effects on gating. Mutations and depolarization (to remove channel block) changed the diliganded gating equilibrium constant only by changing the unliganded gating equilibrium constant (E(0)) and did not alter the energy from ligand binding. All of the tested perturbations were approximately energetically independent. We conclude that naturally occurring mutations mainly adjust E(0) and cause human disease because they generate AChRs that have physiologically inappropriate values of this constant. The results suggest that the energy associated with a structural change of a side chain in the gating isomerization is dissipated locally and is mainly independent of rigid body or normal mode motions of the protein. Gating rate and equilibrium constants are estimated for seven different AChR agonists using a stepwise engineering approach.

Glycine hinges with opposing actions at the acetylcholine receptor-channel transmitter binding site.

The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (α subunit loop B) with regard to both of these parameters. αGly147 is an "activation" hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting conformation for agonists and net ligand binding energy. αGly153 is a "deactivation" hinge that maintains low values for these parameters. αTrp149 (between these two glycines) serves mainly to provide ligand binding energy for gating. We propose that a concerted motion of the two glycine hinges (plus other structural elements at the binding site) positions αTrp149 so that it provides physiologically optimal binding and gating function at the nerve-muscle synapse.

Energetics of gating at the apo-acetylcholine receptor transmitter binding site.

Acetylcholine receptor channels switch between conformations that have a low versus high affinity for the transmitter and conductance for ions (R<-->R*; gating). The forward isomerization, which begins at the transmitter binding sites and propagates approximately 50 A to the narrow region of the pore, occurs by approximately the same sequence of molecular events with or without agonists present at the binding sites. To pinpoint the forces that govern the R versus R* agonist affinity ratio, we measured single-channel activation parameters for apo-receptors having combinations of mutations of 10 transmitter binding site residues in the alpha (Y93, G147, W149, G153, Y190, C192, and Y198), epsilon (W55 and P121), or delta (W57) subunit. Gating energy changes were largest for the tryptophan residues. The alphaW149 energy changes were coupled with those of the other aromatic amino acids. Mutating the aromatic residues to Phe reduces the R/R* equilibrium dissociation constant ratio, with alphaY190 and alphaW149 being the most sensitive positions. Most of the mutations eliminated long-lived spontaneous openings. The results provide a foundation for understanding how ligands trigger protein conformational change.

Energy and structure of the M2 helix in acetylcholine receptor-channel gating.

We studied single-channel currents from neuromuscular acetylcholine receptor-channels with mutations in the pore-lining, M2 helix of the epsilon-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E(2)), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E(2) on a log-log scale (Phi), which illuminates the energy character of the residue (C- versus O-like) within the C<-->O isomerization process; and 3), the open-channel current amplitude (i(0)), which reports whether a mutation alters the energetics of ion permeation. The largest E(2) changes were observed in the cytoplasmic half of epsilonM2 (5', 9', 12', 13', and 16'), with smaller changes apparent for residues > or =17'. Phi was approximately 0.54 for most epsilonM2 residues, but was approximately 0.32 at the positions that had largest E(2) changes. An arginine substitution reduced i(0) significantly at six positions, with the magnitude of the reduction increasing, 16'-->2'. The measurements suggest that the 9', 12', and 13' residues experience large and late free-energy changes in the channel-opening process. We speculate that in the gating isomerization the pore-facing residues >6' and <16' experience multiple energy perturbations associated with changes in protein structure and, perhaps, hydration.

Unliganded gating of acetylcholine receptor channels.

We estimated the unliganded opening and closing rate constants of neuromuscular acetylcholine receptor-channels (AChRs) having mutations that increased the gating equilibrium constant. For some mutant combinations, spontaneous openings occurred in clusters. For 25 different constructs, the unliganded gating equilibrium constant (E(0)) was correlated with the product of the predicted fold-increase in the diliganded gating equilibrium constant caused by each mutation alone. We estimate that (i) E(0) for mouse, wild-type alpha(2)beta delta epsilon AChRs is approximately 1.15 x 10(-7); (ii) unliganded AChRs open for approximately 80 micros, once every approximately 15 min; (iii) the affinity for ACh of the O(pen) conformation is approximately 10 nM, or approximately 15,600 times greater than for the C(losed) conformation; (iv) the ACh-monoliganded gating equilibrium constant is approximately 1.7 x 10(-3); (v) the C-->O isomerization reduces substantially ACh dissociation, but only slightly increases association; and (vi) ACh provides only approximately 0.9 k(B)T more binding energy per site than carbamylcholine but approximately 3.1 k(B)T more than choline, mainly because of a low O conformation affinity. Most mutations of binding site residue alphaW149 increase E(0). We estimate that the mutation alphaW149F reduces the ACh affinity of C only by 13-fold, but of O by 190-fold. Rate-equilibrium free-energy relationships for different regions of the protein show similar slopes (Phi values) for un- vs. diliganded gating, which suggests that the conformational pathway of the gating structural change is fundamentally the same with and without agonists. Agonist binding is a perturbation that (like most mutations) changes the energy, but not the mechanism, of the gating conformational change.

Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap.

Gating of nicotinic acetylcholine receptors from a C(losed) to an O(pen) conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where C<-->O gating motions generate a low<-->high change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the alpha subunit ('alphaM2-cap'). This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. alphaM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three alphaM2-cap residues (alphaI260, alphaP265 and alphaS268) appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the alphaM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.

Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker.

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a "gate" in the transmembrane domain (TMD). We used Phi-value analysis to probe the relative timing of the gating motions of alpha-subunit residues located near the ECD-TMD interface. Mutation of four of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (K(eq)) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Phi-value for the whole linker was approximately 0.64. One interpretation of this result is that the gating motions of the M2-M3 linker are approximately synchronous with those of much of M2 (approximately 0.64), but occur after those of the transmitter binding site region (approximately 0.93) and loops 2 and 7 (approximately 0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed K(eq) by 2800-, 10-, and 18-fold, respectively, and with an average Phi-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (< or =0.51 kcal mol(-1)). The M2-M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an approximately 16-A border and involve about a dozen residues.

Acetylcholine receptor gating: movement in the alpha-subunit extracellular domain.

Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ("Phi blocks") move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the alpha-subunit, the first Phi-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the beta-core of the alpha-subunit. Y127 is at the boundary between the first and second Phi blocks, at a subunit interface. alphaY127 mutations cause large changes in the gating equilibrium constant and with a characteristic Phi-value (Phi = 0.77) that places this residue in the second Phi-block. We also examined the effect on gating of mutations in neighboring residues deltaI43 (Phi = 0.86), epsilonN39 (complex kinetics), alphaI49 (no effect) and in residues that are homologous to alphaY127 on the epsilon, beta, and delta subunits (no effect). The extent to which alphaY127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in alphaY127 (in both alpha subunits) plus residues alphaD97 or deltaI43. The magnitude of the coupling between alphaD97 and alphaY127 depended on the alphaY127 side chain and was small for both H (0.53 kcal/mol) and C (-0.37 kcal/mol) substitutions. The coupling across the single alpha-delta subunit boundary was larger (0.84 kcal/mol). The Phi-value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the first Phi-block and is not synchronous with those of alphaY127. This suggests that the inner and outer sheets of the alpha-subunit beta-core do not rotate as a rigid body.

Acetylcholine receptor gating at extracellular transmembrane domain interface: the "pre-M1" linker.

Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K(eq)), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K(eq) (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Phi values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Phi values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only -0.33 kcal/mol (for both alpha subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

A stepwise mechanism for acetylcholine receptor channel gating.

Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. We used single-molecule kinetic analysis to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the alpha-subunit of the murine acetylcholine receptor. This analysis produces a 'Phi-value' for a given residue that reflects its open-like versus closed-like character at the transition state. Here we show that most of the residues throughout the length of M2 have a Phi-value of approximately 0.64 but that some near the middle have lower Phi-values of 0.52 or 0.31, suggesting that alphaM2 moves in three discrete steps. The core of the channel serves both as a gate that regulates ion flow and as a hub that directs the propagation of the gating isomerization through the membrane domain of the acetylcholine receptor.