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).

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.

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.

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.

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.

Pathways for nicotinic receptor desensitization.

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.

CHRFAM7A: A human specific fusion gene, accounts for the translational gap for cholinergic strategies in Alzheimer's disease.

BACKGROUND: Cholinergic neuronal loss is one of the hallmarks of AD related neurodegeneration; however, preclinical promise of α7 nAChR drugs failed to translate into humans. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of α7 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 Aß 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 α7 nAChR mediated Aß 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.

iPSC model of CHRFAM7A effect on α7 nicotinic acetylcholine receptor function in the human context.

The α7 nicotinic acetylcholine receptor (α7nAChR) has been a promising target for diseases affecting cognition and higher cortical functions; however, the effect observed in animal models failed to translate into human clinical trials identifying a translational gap. CHRFAM7A is a human-specific fusion gene with properties that enable incorporation into the α7nAChR and, being human specific, CHRFAM7A effect was not accounted for in preclinical studies. We hypothesized that CHRFAM7A may account for this translational gap and understanding its function may offer novel insights when exploring α7nAChR as a drug target. CHRFAM7A is present in different copy number variations (CNV) in the human genome with high frequency. To study the functional consequences of the presence of the CHRFAM7A, two induced pluripotent stem cell (iPSC) lines (0 copy and 1 copy direct) were developed. The 0 copy line was rescued with CHRFAM7A transfection to control for genetic heterogeneity. As readouts for genotype-phenotype correlation, α7nAChR synaptic transmission and amyloid beta 1-42 (Aß1-42) uptake were tested. Synaptic transmission in the presence of CHRFAM7A demonstrated that PNU-modulated desensitization of α7nAChR currents increased as a function of CHRFAM7A dosage. CHRFAM7A mitigated the dose response of Aß1-42 uptake suggesting a protective effect beyond physiological concentrations. Furthermore, in the presence of CHRFAM7A Aß1-42 uptake activated neuronal interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) without activating the canonical inflammasome pathway. Lead optimization may identify more potent molecules when the screen has a model harboring CHRFAM7A. Incorporating pharmacogenetics into clinical trials may enhance signals in efficacy measures.

A single molecular distance predicts agonist binding energy in nicotinic receptors.

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.

Efficiency measures the conversion of agonist binding energy into receptor conformational change.

Receptors alternate between resting↔active conformations that bind agonists with low↔high affinity. Here, we define a new agonist attribute, energy efficiency (η), as the fraction of ligand-binding energy converted into the mechanical work of the activation conformational change. η depends only on the resting/active agonist-binding energy ratio. In a plot of activation energy versus binding energy (an "efficiency" plot), the slope gives η and the y intercept gives the receptor's intrinsic activation energy (without agonists; ΔG0). We used single-channel electrophysiology to estimate η for eight different agonists and ΔG0 in human endplate acetylcholine receptors (AChRs). From published equilibrium constants, we also estimated η for agonists of KCa1.1 (BK channels) and muscarinic, γ-aminobutyric acid, glutamate, glycine, and aryl-hydrocarbon receptors, and ΔG0 for all of these except KCa1.1. Regarding AChRs, η is 48-56% for agonists related structurally to acetylcholine but is only ∼39% for agonists related to epibatidine; ΔG0 is 8.4 kcal/mol in adult and 9.6 kcal/mol in fetal receptors. Efficiency plots for all of the above receptors are approximately linear, with η values between 12% and 57% and ΔG0 values between 2 and 12 kcal/mol. Efficiency appears to be a general attribute of agonist action at receptor binding sites that is useful for understanding binding mechanisms, categorizing agonists, and estimating concentration-response relationships.

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.

A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip.

Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing ("gating") between resting and active conformations under the influence of neurotransmitters. Here, to explore the mechanisms that link the transmitter-binding sites (TBSs) with the distant gate, we use mutant cycle analyses to measure coupling between residue pairs, phi value analyses to sequence domain rearrangements, and current simulations to reproduce a microsecond shut component ("flip") apparent in single-channel recordings. Significant interactions between amino acids separated by >15 Å are rare; an exception is between the αM2-M3 linkers and the TBSs that are ∼30 Å apart. Linker residues also make significant, local interactions within and between subunits. Phi value analyses indicate that without agonists, the linker is the first region in the protein to reach the gating transition state. Together, the phi pattern and flip component suggest that a complete, resting↔active allosteric transition involves passage through four brief intermediate states, with brief shut events arising from sojourns in all or a subset. We derive energy landscapes for gating with and without agonists, and propose a structure-based model in which resting→active starts with spontaneous rearrangements of the M2-M3 linkers and TBSs. These conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a "bubble" that collapses to initiate ion conduction. The energy landscapes suggest that twisting is the most energetically unfavorable step in the resting→active conformational change and that the rate-limiting step in the reverse process is bubble formation.

Structural correlates of affinity in fetal versus adult endplate nicotinic receptors.

Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ∼30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

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.

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.

Activation of endplate nicotinic acetylcholine receptors by agonists.

The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ∼300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of α(1) and one each of ß, δ, and either γ (fetal) or ϵ (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αϵ subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C→O opening rate constant and C↔O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly to reach a peak that corresponds to PO ∼0.96.

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.

Agonist activation of a nicotinic acetylcholine receptor.

How does an agonist activate a receptor? In this article I consider the activation process in muscle nicotinic acetylcholine receptors (AChRs), a prototype for understanding the energetics of binding and gating in other ligand-gated ion channels. Just as movements that generate gating currents activate voltage-gated ion channels, movements at binding sites that generate an increase in affinity for the agonist activate ligand-gated ion channels. The main topics are: i) the schemes and intermediate states of AChR activation, ii) the energy changes of each of the steps, iii) the sources of the energies, iv) the three kinds of AChR agonist binding site and v) the correlations between binding and gating energies. The binding process is summarized as sketches of different conformations of an agonist site. The results suggest that agonists lower the free energy of the active conformation of the protein in stages by establishing favorable, local interactions at each binding site, independently. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.