Gating Isomerization Research Articles

Between the Sheets: Inter-Subunit Backbone Interactions at AChR Neurotransmitter Binding Sites

The two neuromuscular AChR agonist-binding pockets are sandwiched between β-structures of α and e/δ/γ subunits. Within the global, channel-opening gating isomerization, a low-to-high affinity change for the agonist at each site serves to increase PO. There are two conserved, vicinal prolines at the end strand β5’ in each non-α subunit. We used single-channel electrophysiology to estimate energy changes caused by mutations here (γP120 and γP121), with regard to unliganded gating and the affinity change. For both prolines, all mutations had little or no effect on constitutive activity in all subunits. AFLR mutations of the first proline had no effect on the affinity change for ACh in e and δ subunits, but a modest effect in γ. At γP121, R and L substitutions had a large effect on the affinity change (>+3kcal/mol) but AGY were off less consequence (for all mutations the effect was smaller for choline than for ACh). Mutant cycle analyses were used to measure free energy coupling regarding the affinity change for ACh between γP121 and other residues near the binding pocket. γP121A interacts strongly with αG147A (strand β7) and αY198A (strand β10), modestly with αW149A (loop B) and αY190A (loop C) and not at all with αY93A (loop A) and αG153A (loop B). There was no coupling between γP121A and its neighbor γW55A (strand β1). The results suggest that strand β5’ in the γ subunit interacts with strands β7 and β10 in the α subunit, but less-so with residues in flexible loops that nonetheless influence affinity. Our working hypothesis is that β strands of the α and non-α subunits exchange energy during the affinity change for the agonist.

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.

Introducing Kappa: An Integrated, ‘Catch-And-Hold’ Reaction Co-Ordinate that Triggers AChR Gating

For many years drug action on membrane receptors has been conceptualized as occurring in two distinct steps, initially termed ‘affinity’ and ‘intrinsic activity’. In ligand-gated ion channels these stages of activation are called ‘binding’ and ‘gating’. Single-channel kinetic analysis of adult-type mouse neuromuscular acetylcholine receptor-channels (AChRs) indicates that a conformational change in the protein is an essential part of the ligand association process, and that this structural rearrangement shares a common mechanism with the ensuing low-to-high affinity transformation of the binding site that triggers channel-gating. The energy source for gating is the change in affinity for the agonist. We find that in AChRs there is a close correlation between the low- and high-affinity equilibrium association constants. This indicates that agonist association and the low-to-high affinity-change that powers gating are linked processes that reflect a single, integrated ‘catch-and-hold’ structural adaptation of the protein to the ligand. The slope of the correlation, kappa, is an index of the relative position in this reaction co-ordinate, which occurs in the initial stages of the gating isomerization. Residue GlyB2 changes energy (‘moves’) early in the process (k=0.85), before the agonist and four a-subunit aromatic amino acids (k≈0.5). In 1957 del Castillo and Katz separated binding and gating. The results suggest that they are just different stages of a single reaction co-ordinate.

Thinking in cycles: MWC is a good model for acetylcholine receptor‐channels

Neuromuscular acetylcholine receptors have long been a model system for understanding the mechanisms of operation of ligand-gated ion channels and fast chemical synapses. These five subunit membrane proteins have two allosteric (transmitter) binding sites and a distant ion channel domain. Occupation of the binding sites by agonist molecules transiently increases the probability that the channel is ion-permeable. Recent experiments show that the Monod, Wyman and Changeux formalism for allosteric proteins, originally developed for haemoglobin, is an excellent model for acetylcholine receptors. By using mutations and single-channel electrophysiology, the gating equilibrium constants for receptors with zero, one or two bound agonist molecules, and the agonist association and dissociation rate constants from both the closed- and open-channel conformations, have been estimated experimentally. The change in affinity for each transmitter molecule between closed and open conformations provides ~-5.1 kcal mol(-1) towards the global gating isomerization of the protein.

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.

Energy Changes at the Acetylcholine Receptor Gate Region

Abstract The neuromuscular acetylcholine receptor (AChR) alternatively switches between an inactive (closed) and active (open) conformation. during the gating isomerization (R↔R∗) process. The ‘gate’ region is formed by the equatorial residues (9’-16’). We and others have speculated that channel gating involves a wetting/de-wetting of this region of the pore, based on measurements of gating equilibrium changes caused by mutations, phi value analysis and molecular dynamics simulations. We are studying the coupling energies between 9’-13’ residues in different subunits, by estimating the effect of mutations, separately vs. in combination, on the unliganded gating equilibrium constant E0. The end states and conformational pathway for AChR gating is fundamentally the same with and without agonists, and E0 can be estimated without obfuscation by channel block. We characterized two background mutants (αA96N/W149S and αA96H/Y96H) that greatly increase spontaneous gating and exhibit simple two-state kinetics (E0 = 0.076 and 0.031). These E0 values predict an E0wt value that is similiar to previous estimates (∼6.5x10−7). With these spontaneously-opening backgrounds, the individual free energy changes for βV13A and δV13'L (−4.1 kcal/mol and −1.9 kcal/mol) are similar to previous estimates obtained by using diliganded AChRs. Initial results suggest that these 13’ mutants are only weakly coupled (∼-1.2 kcal/mol). We expect these studies will help unravel the structural changes in protein and water that occur at the AChR gate region.

Mapping Heat Exchange in an Allosteric Protein

Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein.

Subunit Symmetry at the Extracellular Domain-Transmembrane Domain Interface in Acetylcholine Receptor Channel Gating

Transmitter molecules bind to synaptic acetylcholine receptor channels (AChRs) to promote a global channel-opening conformational change. Although the detailed mechanism that links ligand binding and channel gating is uncertain, the energy changes caused by mutations appear to be more symmetrical between subunits in the transmembrane domain compared with the extracellular domain. The only covalent connection between these domains is the pre-M1 linker, a stretch of five amino acids that joins strand β10 with the M1 helix. In each subunit, this linker has a central Arg (Arg(3')), which only in the non-α-subunits is flanked by positively charged residues. Previous studies showed that mutations of Arg(3') in the α-subunit alter the gating equilibrium constant and reduce channel expression. We recorded single-channel currents and estimated the gating rate and equilibrium constants of adult mouse AChRs with mutations at the pre-M1 linker and the nearby residue Glu(45) in non-α-subunits. In all subunits, mutations of Arg(3') had similar effects as in the α-subunit. In the ε-subunit, mutations of the flanking residues and Glu(45) had only small effects, and there was no energy coupling between εGlu(45) and εArg(3'). The non-α-subunit Arg(3') residues had Φ-values that were similar to those for the α-subunit. The results suggest that there is a general symmetry between the AChR subunits during gating isomerization in this linker and that the central Arg is involved in expression more so than gating. The energy transfer through the AChR during gating appears to mainly involve Glu(45), but only in the α-subunits.

Linking the Acetylcholine Receptor-Channel Agonist-Binding Sites with the Gate

The gating isomerization of neuromuscular acetylcholine receptors links the rearrangements of atoms at two transmitter-binding sites with those at a distant gate region in the pore. To explore the mechanism of this reversible process, we estimated the gating rate and equilibrium constants for receptors with point mutations of α-subunit residues located between the binding sites and the membrane domain (N95, A96, Y127, and I49). The maximum energy change caused by a side-chain substitution at αA96 was huge (∼8.6 kcal/mol, the largest value measured so far for any α-subunit amino acid). A Φ-value analysis suggests that αA96 experiences its change in energy (structure) approximately synchronously with residues αY127 and αI49, but after the agonist molecule and other residues in loop A. Double mutant-cycle experiments show that the energy changes at αA96 are strongly coupled with those of αY127 and αI49. We identify a column of mutation-sensitive residues in the α-subunit that may be a pathway for energy transfer through the extracellular domain in the gating isomerization.

Acetylcholine Receptor Channels Activated by a Single Agonist Molecule

The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R↔R∗). The R∗ shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded gating equilibrium constant E1 and the energy change associated with the R versus R∗ change in affinity for agonists. AChRs with only one operational binding site gave rise to a single population of currents, indicating that the two transmitter binding sites have approximately the same affinity for the transmitter ACh. The results indicated that E1 ≈ 4.3 × 10−3 with ACh, and ≈1.7 × 10−4 with the partial-agonist choline. From these values and the diliganded gating equilibrium constants, we estimate that the unliganded AChR gating constant is E0 ≈ 6.5 × 10−7. Gating changes the stability of the ligand-protein complex by ∼5.2 kcal/mol for ACh and ∼3.3 kcal/mol for choline.

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 ɛ-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E2), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E2 on a log-log scale (Φ), 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 (i0), which reports whether a mutation alters the energetics of ion permeation. The largest E2 changes were observed in the cytoplasmic half of ɛM2 (5′, 9′, 12′, 13′, and 16′), with smaller changes apparent for residues ≥17′. Φ was ∼0.54 for most ɛM2 residues, but was ∼0.32 at the positions that had largest E2 changes. An arginine substitution reduced i0 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.

Temperature Dependence And Activation Energy of nAChR Gating

Neuromuscular acetylcholine receptors (AChRs) are ion channels that alternately adopt conformations that either allow or prohibit the flow of ions across the membrane. These two stable end states are separated by an energy barrier, the peak of which is called the transition region. The energy of the transition region relative to the end states is the reaction activation energy (Eact). To quantify Eact, we studied the temperature dependence of single-channel gating rate constants (ko, opening; kc, closing) for wt and mutant AChRs, activated by different ligands or without any added ligand, over a range of temperatures (5-35 oC, HEK cells, cell attached, -110 mV, mouse α2βδe). The results were fitted by the Arrhenius equation: k(T)=A∗exp (-Eact/RT). Increasing the temperature from 50 C to 350 C increased kc for wt and δL265T AChRs activated by choline, each by ∼35-fold (Eact=20.5 and 23.6 kcal/mol, respectively). We also estimated the temperature sensitivity of ko and kc in four more constructs with one or more point mutations in both α subunits. For all four, ko and kc increased with temperature: Eact (ko and kc; kcal/mol)=21 and 23 (Y127E activated by ACh); 24 and 27 (D97A + Y127F + S269I, unliganded); 29 and 23 (D97A + Y127F + S269I + W149F, unliganded); 29 and 25 (G153S activated by choline). For these six constructs the average activation energy was ∼24.6 kcal/mol for both closing and opening. This quantity did not change with the agonist (including water) or the mutations. This suggests that the energy barrier for the gating isomerization is not significantly determined by the ligands at the transmitter binding sites or by the gating motions of the mutated residues, and that unliganded and diliganded AChR gating likely proceed by similar reaction pathways.

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.

Dynamics of the acetylcholine receptor pore at the gating transition state.

Neuromuscular acetylcholine receptors (AChRs) are ion channels that alternatively adopt stable conformations that either allow (open) or prohibit (closed) ionic conduction. We probed the dynamics of pore (M2) residues at the diliganded gating transition state by using single-channel kinetic and rate-equilibrium free energy relationship (phi-value) analyses of mutant AChRs. The mutations were at the equatorial (9') position of the alpha, beta, and epsilon subunits (n = 15) or at sites between the equator and the extracellular domain in the alpha-subunit (n = 8). We also studied AChRs having only one of the two alpha-subunits mutated. The results indicate that the alpha-subunit, like the delta-subunit, has a region of flexure near the middle of M2, that the two alpha-subunits experience distinct energy barriers to gating at the equator (but not elsewhere), and that the collective subunit motions at the equator are asymmetric during the AChR gating isomerization.