BackgroundHuman 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.MethodsTwo 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.FindingsCHRFAM7A/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.InterpretationCHRFAM7A 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.FundingThis work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

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.

Agonists turn on receptors because they provide net favorable binding energy to active versus resting conformations of their target sites. We used simulations to explore conformational dynamics of the weak→strong binding transition at the Torpedo α–δ nicotinic acetylcholine receptor orthosteric site. Using 4 agonists, the alternative site conformations were identified in trajectories generated from a single starting structure by matching binding energies calculated in silico with those measured experimentally in vitro . The weak→strong transition starts with a rotation of the agonist about its cationic center (‘flip’), followed by a downward displacement of loop C that repositions αY190 (‘flop’), followed by formation of H-bonds between the ligand, a structural water and the δ subunit loop E backbone (‘fix’). The result is a compact, hydrophobic and stable pocket with higher affinity for agonists. The simulations reveal a transient intermediate state in the weak→strong transition.

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.

Receptors switch between resting-active conformations (C-O) under the influence of agonists that bind weakly versus strongly. We estimated the two equilibrium dissociation constants, KdC and KdO, from single channel currents of adult neuromuscular nicotinic acetylcholine receptors (AChRs) either by kinetic modeling or from dose-response curves. After removing the chemical potential by normalizing with constants from a standard agonist, the efficiency (η) at which chemical binding energy is converted into mechanical work of gating was calculated as η=1-κ where κ is the free energy ratio logKdC/logKdO. η is also a measure of the correlation between agonist efficacy and affinity. In wt AChRs, ηACh is 50% at the adult α-ε and α-δ neurotransmitter sites, and 56% at fetal α-γ. We measured η for 22 agonists in wt AChRs or for several agonists after a mutation of 8 different binding site amino acids. With both types of perturbation there are 6 η-classes (30, 41, 45, 51, 54 and 60%), with smaller agonists generally having a greater efficiency. Elsewhere we show that for 4 agonists, η values calculated from structures by using MD simulations agree with those obtained experimentally. Efficiency is a universal agonist attribute that offers a new way to classify ligands and a window into the structural basis of receptor activation by agonists.

ABSTRACT Receptors signal by switching between resting and active shapes under the influence of agonists. The maximum response produced by an agonist (‘efficacy’) depends on its relative binding strength (‘affinity’) to active versus resting conformations. Efficiency, the logcorrelation between these two agonist properties, is the fraction of binding energy converted into energy for the receptor’s conformational change. In adult muscle nicotinic receptors, efficiencies estimated from 76 concentration-response curves (23 agonists, 53 mutations) segregate into 5 discrete classes (%): 0.56 (17), 0.51(32), 0.45(13), 0.41(26) and 0.31(12). There is a strong linear correlation between affinity and efficacy within each class, but the multiplicity of classes precludes the appearance of any overall relationship. The efficiency class distribution indicates that there are at least 5 resting versus active binding site structural pairs. We discuss efficiency as a quantitative measure of energy coupling between agonist binding and protein conformational change that is fundamental to receptor operation.

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.

Agonists turn on nicotinic acetylcholine receptors (AChRs) because they bind weakly to resting-C and strongly to active-O conformations of their target sites. Agonist efficiency (η) is the percent of chemical binding energy converted into the mechanical work of C-O gating that depends on the weak/strong binding energy ratio, η=1-ΔΔGC/ΔΔGO. κ has been measured in adult muscle nicotinic AChRs for many agonists and binding site mutations by using electrophysiology. We have compared these experimental values to those calculated from binding free energy estimates to C versus O states at the α-δ binding site. MD simulations were done with an agonist bound to the neurotransmitter site of a muscle AChR (6UVW.pdb). After removing the bound Btx, ligands were docked into the site of the extracellular domain dimer. A five-step equilibration (pre-production) was followed by 200 ns of un-restrained MD simulation using AMBER 2019. The three energy minimas was identified in sequence (m1, m2 and m3) using Principle component analysis (PCA), furthermore their free energies were calculated via MM-PBSA. In general the calculated binding free energy didn’t corroborate well with experimental ones, the calculated binding free energy ratios (η) values for epibatidine, epiboxidine, carbamylcholine and ACh were similar to those estimated by using electophysiology. The results confirm the correctness of our identification of closed-C (m1) versus open-O (m3) binding pocket structures, and suggest that it might be possible in general to calculate agonist efficiency using crystal and MD studies. The structural correlates of agonist efficiency are under investigation.

Agonists are evaluated by a concentration-response curve (CRC), with a midpoint (EC50) that indicates potency and a high-concentration asymptote (Pomax) that indicates efficacy. The low-concentration asymptote indicates constitutive activity and is agonist-independent. A third agonist attribute, efficiency (η eta) 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 for muscle nicotinic acetylcholine receptors (AChR) derived from either single-channel or whole-cell responses. For 20 agonists the distribution of η values is multimodal with population means at 55% (including TMA), 51% (including ACh, nornicotine and DMPP), 42% (including epibatidine and cytisine) and possibly 35% (including varenicline). Agonist potency and efficacy are related by η, which otherwise appear to be unrelated. The value of η decreases with increasing volume of the agonist's head-group. Many binding site mutations have only a small effect on ACh efficiency except for αY190A (35%), αW149A (60%) and those at αG153 (42%, including one that causes CMS). αD200A decreased the efficiency more for epibatidine (29%) compared to ACh (37%), giving an insight into the mechanistic differences into the way these agonists act. If η is known, EC50 and Pomax 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 CRCs that have been normalized to 1. Given η, the level of constitutive activity can be estimated from a single CRC. Efficiency is the missing link between affinity and efficacy.

Concentration-response curves (CRCs) are characterized by a midpoint (EC50) and a high-concentration asymptote (POmax) that relate to the agonist's affinity and efficacy. Recently, a third agonist attribute, efficiency, was defined as the fraction of ligand binding energy that is applied to the conformational change that activates the receptor. We show that efficiency can be calculated from EC50 and POmax and estimate its value for 17 agonists of a nicotinic acetylcholine receptor (AChR). Adult skeletal muscle AChRs were expressed in HEK cells and CRCs were compiled from single-channel currents to estimate EC50 and POmax. There were two populations of agonists having an efficiency of 52±2% (n=11) or 41±3% (n=6) (mean±s.d). Agonists with a smaller head-group volume (70±9 A3) have a higher efficiency than those with a larger volume (102±18 A3). Examples of agonists in the high-efficiency/small-volume group include quaternary amines (ACh, carbamylcholine, tetramethyammonium and others) as well as secondary amines (nornicotine, anabaesine and DMPP). Low-efficiency/large-volume agonists include smoking-cessation drugs (varenicline and cytosine), cations (tetraethlyammonium and tetramethylphosphonium) and secondary amines (epibatidine, anatoxin). αY190A is the only one of 22 binding site mutations that affect the efficiency of ACh, switching it from the higher- to the lower-efficiency population. If agonist efficiency is known, EC50 can be estimated from POmax and the receptor's constitutive level of activity can be calculated from any CRC.

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.

ABSTRACTAgonists are classified by the strength at which they bind to their target sites (affinity) and their ability to activate receptors once bound to those sites (efficacy). Efficiency is a third fundamental agonist property that is a measure of the correlation between affinity and efficacy. Efficiency is the percent of agonist binding energy that is converted into energy for receptor activation (‘gating’). In the muscle nicotinic acetylcholine receptor, agonists belong to families having discrete efficiencies of 54%, 51%, 42% or 35%. Efficiency depends on the size and composition of both the agonist and binding site, and can be estimated from, and used to interpret, concentration-response curves. A correlation between affinity and efficacy indicates that the agonist’s energy changes that take place within binding and gating processes are linked. Efficiency suggests that receptors turn on and off by progressing through a sequence of energy-linked domain rearrangements, as in a zipper.

Nicotinic acetylcholine receptors (AChRs) are ligand-gated ion channels that generate transient currents by binding agonists and switching rapidly between closed- and open-channel conformations. Upon sustained exposure to ACh, the cell response diminishes slowly because of desensitization, a process that shuts the channel even with agonists still bound. In liganded receptors, the main desensitization pathway is from the open-channel conformation, but after agonists dissociate the main recovery pathway is to the closed-channel conformation. In this Viewpoint, I discuss two mechanisms that can explain the selection of different pathways, a question that has puzzled the community for 60 yr. The first is based on a discrete-state model (the “prism”), in which closed, open, and desensitized conformational states interconnect directly. This model predicts that 5% of unliganded AChRs are desensitized. Different pathways are taken with versus without agonists because ligands have different energy properties (φ values) at the transition states of the desensitization and recovery reactions. The second is a potential energy surface model (the “monkey saddle”), in which the states connect indirectly at a shared transition state region. Different pathways are taken because agonists shift the position of the gating transition state relative to the point where gating and desensitization conformational trajectories intersect. Understanding desensitization pathways appears to be a problem of kinetics rather than of thermodynamics. Other aspects of the two mechanisms are considered, as are experiments that may someday distinguish them.

Nicotinic acetylcholine receptors (AChRs) enter high-affinity, non-conducting, ‘desensitized’ (D) conformations upon sustained exposure to high concentrations of agonists. Desensitized conformations resemble functionally both resting (R; low-affinity, non-conducting) and active (R∗; high-affinity, ion-conducting) states. In the presence of agonists entry into D state(s) is mainly from R∗, but upon agonist washout recovery is mainly into R. Franke et al. estimated the probability of opening during recovery (PO,R; passing through R∗) in WT mouse neuromuscular AChRs to be ∼10E-2. We used gain-of-function mutations that do not affect agonist binding to monitor channel activity during recovery because they boost constitutive activity. The probability density function of openings during recovery from application of saturating ACh (100 μM for 60 sec) to outside-out patches showed slow (seconds) recovery. Quantitative comparison of terminal PO,G in epochs preceding ACh application correlated well with terminal PO,R obtained from epochs succeeding stimulation. We demonstrate that PO,R is equal to the PO of constitutive gating (PO,G of 7.4x10E-7), which implies that WT receptors rarely recover directly to R∗. Desensitized channels do not appear to recover via an intermediate in the transition state ensemble; this result however, is limited by our inability to discriminate PO,G- from PO,R-openings.

Background: Cholinergic neuronal loss is one of the hallmarks of AD related neurodegeneration; however, preclinical promise of alpha 7 nAChR drugs failed to translate into humans. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of alpha 7 nAChR and was unaccounted for in preclinical models. Methods: Molecular methods: Function of CHRFAM7A alleles was studied in vitro in two disease relevant phenotypic readouts: electrophysiology and A beta uptake. Genome edited human induced pluripotent stem cells (iPSC) were used as a model system with the human context. Double blind pharmacogenetic study: We performed double-blind pharmacogenetic analysis on the effect of AChEI therapy based on CHRFAM7A carrier status in two paradigms: response to drug initiation and DMT effect. Mini Mental Status Examination (MMSE) was used as outcome measure. Change in MMSE score from baseline was compared by 2-tailed T-test. Longitudinal analysis of clinical outcome (MMSE) was performed using a fitted general linear model, based on an assumed autoregressive covariance structure. Model independent variables included age, sex, and medication regimen at the time of the first utilized outcome measure (AChEI alone or AChEI plus memantine), APOE4 carrier status (0, 1 or 2 alleles as categorical variables) and CHRFAM7A genotype. Findings: The direct and inverted alleles have distinct phenotypes. Functional CHRFAM7A allele classifies the population as 25% non-carriers and 75% carriers. Induced pluripotent stem cell (iPSC) models alpha 7 nAChR mediated A beta neurotoxicity. Pharmacological readout translates into both first exposure (p = 0.037) and disease modifying effect (p = 0.0048) in two double blind pharmacogenetic studies. Interpretation: CHRFAM7A accounts for the translational gap in cholinergic strategies in AD. Clinical trials not accounting for this uniquely human genetic factor may have rejected drug candidates that would benefit 25% of AD. Reanalyses of the completed trials using this pharmacogenetic paradigm may identify effective therapy. (C) 2020 The Author(s). Published by Elsevier B.V.

Agonists turn on receptors because they bind more strongly to active (R*) versus resting (R) conformations of their target sites. Here, to explore how agonists activate neuromuscular acetylcholine receptors, we built homology models of R and R* neurotransmitter binding sites, docked ligands to those sites, ran molecular dynamics simulations to relax ("equilibrate") the structures, measured binding site structural parameters, and correlated them with experimental agonist binding energies. Each binding pocket is a pyramid formed by five aromatic amino acids and covered partially by loop C. We found that in R* versus R, loop C is displaced outward, the pocket is smaller and skewed, the agonist orientation is reversed, and a key nitrogen atom in the agonist is closer to the pocket center (distance dx) and a tryptophan pair but farther from αY190. Of these differences, the change in dx shows the largest correlation with experimental binding energy and provides a good estimate of agonist affinity, efficacy, and efficiency. Indeed, concentration-response curves can be calculated from just dx values. The contraction and twist of the binding pocket upon activation resemble gating rearrangements of the extracellular domain of related receptors at a smaller scale.

Nicotinic (endplate) acetylcholine receptors (AChRs) are heteropentamers that alternate between resting⇌active structures (R⇌R∗) that have a low⇌high affinity for agonists and a closed⇆open ion channel. These allosteric proteins are molecular machines that convert chemical energy of ligand binding into kineticenergy of a global change in conformation ('gating'). Previously, single-channel electrophysiology experiments showed that for a series of 10 agonists related structurally to the neurotransmitter ACh, the free energy of binding to R was always approximately half that to R∗. We show that the R/R∗ binding energy ratio determines the binding→gating energy conversion efficiency. Our immediate goal was to understand the physical basis for the increase in affinity (favorable binding energy) that turns on the AChR. We built homology models of R and R∗ endplate AChR extracellular domains, docked ligands and equilibrated by using molecular dynamics simulations. Structural parameters were measured for R and R∗ binding sites (α-δ/ε/γ) occupied by a quaternary amine agonist (ACh, TMA, CCh or choline). In R∗, the agonist binding pocket has a smaller volume and is rotated anti-clockwise, the ligand has a reversed orientation and is closer to the pocket center (distance dx), and loop C is displaced outward. Of 8 structural parameters that differed between R and R∗ conformations, dx was the most-correlated with experimentally-measured binding energy and could account approximately for all agonist actions (affinity, efficacy and efficiency). Indeed, accurate dose-response curves could be calculated from dx values alone. The contraction and rotation of the binding pocket upon activation resemble at small-scale gating rearrangements of the extracellular domain of related receptors and, hence, could be the trigger for the full gating transition of the receptor.

Endplate acetylcholine receptors toggle between resting (R) and active (R∗) conformations. The main connection between these states is through binding agonist molecules. The activation pathway starts with an agonist (A) arriving at a binding site by diffusion, and continues when the agonist enters a cavity lined with aromatic side chains. This process, R→AR ('catch'), forms a low-affinity protein-ligand complex via a local conformation change. Thereafter, a global 'gating' conformation change forms a high-affinity, open-channel complex (AR→AR∗;'hold'), by a process that requires another local rearrangement. To further understand the 'catch' mechanism, we built homology models of R/R∗ (apo) and AR/AR∗ conformations based on crystal structures of AChBP/a4b2, and equilibrated the structures by MD simulations. The results suggest that R is more flexible and has a larger binding pocket that is more-covered by loop C, compared to AR. This is consistent with our hypothesis that loopC acts as a protective lid that is actively opened by the agonist in order to gain entry into the aromatic pocket. Regarding 'hold', previous work showed that the low/high binding energy ratio is constant for a series of agonists structurally-related to ACh, and that this ratio is related to agonist efficiency (the fraction of binding energy converted to gating energy). Further, the efficiency of ACh-like ligands (∼50%) is correlated with the ratio of the distances between the quaternary nitrogen atom and the pocket center. We have extended these studies to include azabicycloamine agonists (related structurally to epibatidine) that have a lower efficiency (∼39%). Preliminary results suggest that for these ligands, too, the distance between the bridge nitrogen atom and the cavity center determines affinity, efficacy and efficiency. We are able to calculate complete concentration-response curves from measurements of these distances in equilibrated structures. NS064969.