Dynamics of receptor activation by agonists.

How do agonists turn on receptors? The model system we have used to address this question is the adult-type skeletal muscle nicotinic acetylcholine receptor. This ligand-gated ion channel has two orthosteric sites (for neurotransmitters) in the extracellular domain linked to an allosteric site (a gate) in the transmembrane domain. The goal of this perspective is to summarize how measurements of agonist binding energy reveal the dynamics of the neurotransmitter sites and the fundamental link between binding and gating.

Agonist efficiency from concentration-response curves: Structural implications and applications.

Agonists are evaluated by a concentration-response curve (CRC), with a midpoint (EC50) that indicates potency, a high-concentration asymptote that indicates efficacy, and a low-concentration asymptote that indicates constitutive activity. A third agonist attribute, efficiency (η), is the fraction of binding energy that is applied to the conformational change that activates the receptor. We show that η can be calculated from EC50 and the asymptotes of a CRC derived from either single-channel or whole-cell responses. For 20 agonists of skeletal muscle nicotinic receptors, the distribution of η-values is bimodal with population means at 51% (including acetylcholine, nornicotine, and dimethylphenylpiperazinium) and 40% (including epibatidine, varenicline, and cytisine). The value of η is related inversely to the size of the agonist's headgroup, with high- versus low-efficiency ligands having an average volume of 70 vs. 102 Å3. Most binding site mutations have only a small effect on acetylcholine efficiency, except for αY190A (35%), αW149A (60%), and those at αG153 (42%). If η is known, the EC50 and high-concentration asymptote can be calculated from each other. Hence, an entire CRC can be estimated from the response to a single agonist concentration, and efficacy can be estimated from EC50 of a CRC that has been normalized to 1. Given η, the level of constitutive activity can be estimated from a single CRC.

Cyclic activation of endplate acetylcholine receptors.

Agonists turn on receptors because they have a higher affinity for active versus resting conformations of the protein. Activation can occur by either of two pathways that connect to form a cycle: Agonists bind to resting receptors that then become active, or resting receptors activate and then bind agonists. We used mutations to construct endplate acetylcholine receptors (AChRs) having only one functional neurotransmitter-binding site and single-channel electrophysiology to measure independently binding constants for four different agonists, to both resting and active conformations of each site. For all agonists and sites, the total free energy change in each pathway was the same, confirming the activation cycle without external energy. Other results show that (i) there is no cooperativity between sites; (ii) agonist association is slower than diffusion in resting receptors but nearly diffusional in active receptors; (iii) whereas resting affinity is determined mainly by agonist association, active affinity is determined mainly by agonist dissociation; and (iv) at each site and for all agonists, receptor activation approximately doubles the agonist-binding free energy. We discuss a two-step mechanism for binding that involves diffusion and a local conformational change ("catch") that is modulated by receptor activation. The results suggest that binding to a resting site and the switch to high affinity are both integral parts of a single allosteric transition. We hypothesize that catch ensures proper signal recognition in complex chemical environments and that binding site compaction is a determinant of both resting and active affinity.

Molecular recognition at cholinergic synapses: acetylcholine versus choline.

KEY POINTS: Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and a low affinity for its metabolic product choline. At each transmitter binding site three aromatic groups determine affinity, and together provide ∼50% more binding energy for ACh than for choline. Deprotonation of αY190 by a nearby lysine strengthens the interaction between this aromatic ring and both ACh and choline. H-bonds position ACh and choline differently in the aromatic cage to generate the different affinities. ABSTRACT: Acetylcholine (ACh) released at the vertebrate nerve-muscle synapse is hydrolysed rapidly to choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally related ligands. To understand how these receptors distinguish ACh and Cho, we used single-channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult-type mouse AChRs having a mutation(s) at the transmitter-binding sites. The aromatic rings of αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist-independent loss in binding energy that depended on the presence of αK145. The results suggest that (1) αY190 is deprotonated by αK145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and (2) AChRs respond strongly to ACh because an H-bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H-bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus Cho binding energies is determined by different ligand positions within a fixed protein structure.

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors.

A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔGB1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔGB1 is ∼-2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔGB1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔGB1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.

Dose-Response Analysis When There Is a Correlation between Affinity and Efficacy.

The shape of a concentration-response curve (CRC) is determined by underlying equilibrium constants for agonist binding and receptor conformational change. Typically, agonists are characterized by the empirical CRC parameters efficacy (the maximum response), EC(50) (the concentration that produces a half-maximum response), and the Hill coefficient (the maximum slope of the response). Ligands activate receptors because they bind with higher affinity to the active versus resting conformation, and in skeletal muscle nicotinic acetylcholine receptors there is an exponential relationship between these two equilibrium dissociation constants. Consequently, knowledge of two receptor-specific, agonist-independent constants--the activation equilibrium constant without agonists (E(0)) and the affinity-correlation exponent (M)--allows an entire CRC to be calculated from a measurement of either efficacy or affinity. I describe methods for estimating the CRCs of partial agonists in receptors that have a correlation between affinity and efficacy.

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

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

Function of 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.

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.

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.

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).

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.

Mechanism of hydrophobic gating in the acetylcholine receptor channel pore.

Neuromuscular acetylcholine receptors (AChRs) are hetero-pentameric, ligand-gated ion channels. The binding of the neurotransmitter acetylcholine (ACh) to two target sites promotes a global conformational change of the receptor that opens the channel and allows ion conduction through the channel pore. Here, by measuring free-energy changes from single-channel current recordings and using molecular dynamics simulations, we elucidate how a constricted hydrophobic region acts as a "gate" to regulate the channel opening in the pore of AChRs. Mutations of gate residues, including those implicated in congenital myasthenia syndrome, lower the permeation barrier of the channel substantially and increase the unliganded gating equilibrium constant (constitutive channel openings). Correlations between hydrophobicity and the observed free-energy changes, supported by calculations of water densities in the wild-type versus mutant channel pores, provide evidence for hydrophobic wetting-dewetting transition at the gate. The analysis of a coupled interaction network provides insight into the molecular mechanism of closed- versus open-state conformational changes at the gate. Studies of the transition state by "phi"(φ)-value analysis indicate that agonist binding serves to stabilize both the transition and the open state. Intersubunit interaction energy measurements and molecular dynamics simulations suggest that channel opening involves tilting of the pore-lining M2 helices, asymmetric outward rotation of amino acid side chains, and wetting transition of the gate region that lowers the barrier to ion permeation and stabilizes the channel open conformation. Our work provides new insight into the hydrophobic gate opening and shows why the gate mutations result in constitutive AChR channel activity.

CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization.

BACKGROUND: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. METHODS: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. FINDINGS: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. INTERPRETATION: CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Energy for wild-type acetylcholine receptor channel gating from different choline derivatives.

Agonists, including the neurotransmitter acetylcholine (ACh), bind at two sites in the neuromuscular ACh receptor channel (AChR) to promote a reversible, global change in protein conformation that regulates the flow of ions across the muscle cell membrane. In the synaptic cleft, ACh is hydrolyzed to acetate and choline. Replacement of the transmitter's ester acetyl group with a hydroxyl (ACh→choline) results in a + 1.8 kcal/mol reduction in the energy for gating generated by each agonist molecule from a low- to high-affinity change of the transmitter binding site (ΔG(B)). To understand the distinct actions of structurally related agonist molecules, we measured ΔG(B) for 10 related choline derivatives. Replacing the hydroxyl group of choline with different substituents, such as hydrogen, chloride, methyl, or amine, increased the energy for gating (i.e., it made ΔG(B) more negative relative to choline). Extending the ethyl hydroxide tail of choline to propyl and butyl hydroxide also increased this energy. Our findings reveal the amount of energy that is available for the AChR conformational change provided by different, structurally related agonists. We speculate that a hydrogen bond between the choline hydroxyl and the backbone carbonyl of αW149 positions this agonist's quaternary ammonium group so as to reduce the cation-π interaction between this moiety and the aromatic groups at the binding site.

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.

Agonist efficiency links binding and gating in a nicotinic receptor.

Receptors signal by switching between resting (C) and active (O) shapes ('gating') under the influence of agonists. The receptor's maximum response depends on the difference in agonist binding energy, O minus C. In nicotinic receptors, efficiency (η) represents the fraction of agonist binding energy applied to a local rearrangement (an induced fit) that initiates gating. In this receptor, free energy changes in gating and binding can be interchanged by the conversion factor η. Efficiencies estimated from concentration-response curves (23 agonists, 53 mutations) sort into five discrete classes (%): 0.56 (17), 0.51(32), 0.45(13), 0.41(26), and 0.31(12), implying that there are 5 C versus O binding site structural pairs. Within each class efficacy and affinity are corelated linearly, but multiple classes hide this relationship. η unites agonist binding with receptor gating and calibrates one link in a chain of coupled domain rearrangements that comprises the allosteric transition of the protein.

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.

The energetic consequences of loop 9 gating motions in acetylcholine receptor-channels.

Acetylcholine receptor-channels (AChRs) mediate fast synaptic transmission between nerve and muscle. In order to better-understand the mechanism by which this protein assembles and isomerizes between closed- and open-channel conformations we measured changes in the diliganded gating equilibrium constant (E(2)) consequent to mutations of residues at the C-terminus of loop 9 (L9) in the α and ε subunits of mouse neuromuscular AChRs. These amino acids are close to two interesting interfaces, between the extracellular and transmembrane domain within a subunit (E­T interface) and between primary and complementary subunits (P­C interface). Most α subunit mutations modestly decreased E(2) (mainly by slowing the channel-opening rate constant) and sometimes produced AChRs that had heterogeneous gating kinetic properties. Mutations in the ε subunit had a larger effect and could either increase or decrease E(2), but did not induce kinetic heterogeneity. There are broad-but-weak energetic interactions between αL9 residues and others at the αE­T interface, as well as between the εL9 residue and others at the P­C interface (in particular, the M2­M3 linker). These interactions serve, in part, to maintain the structural integrity of the AChR assembly at the E­T interface. Overall, the energy changes of L9 residues are significant but smaller than in other regions of the protein.

Decrypting the sequence of structural events during the gating transition of pentameric ligand-gated ion channels based on an interpolated elastic network model.

Despite many experimental and computational studies of the gating transition of pentameric ligand-gated ion channels (pLGICs), the structural basis of how ligand binding couples to channel gating remains unknown. By using a newly developed interpolated elastic network model (iENM), we have attempted to compute a likely transition pathway from the closed- to the open-channel conformation of pLGICs as captured by the crystal structures of two prokaryotic pLGICs. The iENM pathway predicts a sequence of structural events that begins at the ligand-binding loops and is followed by the displacements of two key loops (loop 2 and loop 7) at the interface between the extracellular and transmembrane domain, the tilting/bending of the pore-lining M2 helix, and subsequent movements of M4, M3 and M1 helices in the transmembrane domain. The predicted order of structural events is in broad agreement with the Φ-value analysis of α subunit of nicotinic acetylcholine receptor mutants, which supports a conserved core mechanism for ligand-gated channel opening in pLGICs. Further perturbation analysis has supported the critical role of certain intra-subunit and inter-subunit interactions in dictating the above sequence of events.