Ion transport in muscle acetylcholine receptor maintained by conserved salt bridges between the pore and lipid membrane.

Pores through ion channels rapidly transport small inorganic ions along their electrochemical gradients. Here, applying single-channel electrophysiology and mutagenesis to the archetypal muscle nicotinic acetylcholine receptor (AChR) channel, we show that a conserved pore-peripheral salt bridge partners with those in the other subunits to regulate ion transport. Disrupting the salt bridges in all five receptor subunits greatly decreases the amplitude of the unitary current and increases its fluctuations. However, disrupting individual salt bridges has unequal effects that depend on the structural status of the other salt bridges. The AChR ε- and δ-subunits are structurally unique in harboring a putative palmitoylation site near each salt bridge and bordering the lipid membrane. The effects of disrupting the palmitoylation sites mirror those of disrupting the salt bridges, but the effect of disrupting either of these structures depends on the structural status of the other. Thus, rapid ion transport through the AChR channel is maintained by functionally interdependent salt bridges linking the pore to the lipid membrane.

Pathogenic residue insertion in neuronal nicotinic receptor alters intra- and inter-subunit interactions that tune channel gating.

We describe molecular-level functional changes in the α4ß2 nicotinic acetylcholine receptor by a leucine residue insertion in the M2 transmembrane domain of the α4 subunit associated with sleep-related hyperkinetic epilepsy. Measurements of agonist-elicited single-channel currents reveal the primary effect is to stabilize the open channel state, while the secondary effect is to promote reopening of the channel. These dual effects prolong the durations of bursts of channel openings equally for the two major stoichiometric forms of the receptor, (α4)2(ß2)3 and (α4)3(ß2)2, indicating the functional impact is independent of mutant copy number per receptor. Altering the location of the residue insertion within M2 shows that functionally pivotal structures are confined to a half turn of the M2 α-helix. Residue substitutions within M2 and surrounding α-helices reveal that both intrasubunit and intersubunit interactions mediate the increase in burst duration. These interactions impacting burst duration depend linearly on the size and hydrophobicity of the substituting residue. Together, the results reveal a novel structural region of the α4ß2 nicotinic acetylcholine receptor in which interhelical interactions tune the stability of the open channel state.

Structural bases for stoichiometry-selective calcium potentiation of a neuronal nicotinic receptor.

BACKGROUND AND PURPOSE: α4ß2 nicotinic acetylcholine (nACh) receptors assemble in two stoichiometric forms, one of which is potentiated by calcium. The sites of calcium binding that underpin potentiation are not known. EXPERIMENTAL APPROACH: To identify calcium binding sites, we applied cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations to each stoichiometric form of the α4ß2 nACh receptor in the presence of calcium ions. To test whether the identified calcium sites are linked to potentiation, we generated mutants of anionic residues at the sites, expressed wild type and mutant receptors in clonal mammalian fibroblasts, and recorded ACh-elicited single-channel currents with or without calcium. KEY RESULTS: Both cryo-EM and MD simulations show calcium bound to a site between the extracellular and transmembrane domains of each α4 subunit (ECD-TMD site). Substituting alanine for anionic residues at the ECD-TMD site abolishes stoichiometry-selective calcium potentiation, as monitored by single-channel patch clamp electrophysiology. Additionally, MD simulation reveals calcium association at subunit interfaces within the extracellular domain. Substituting alanine for anionic residues at the ECD sites reduces or abolishes stoichiometry-selective calcium potentiation. CONCLUSIONS AND IMPLICATIONS: Stoichiometry-selective calcium potentiation of the α4ß2 nACh receptor is achieved by calcium association with topographically distinct sites framed by anionic residues within the α4 subunit and between the α4 and ß2 subunits. Stoichiometry-selective calcium potentiation could result from the greater number of calcium sites in the stoichiometric form with three rather than two α4 subunits. The results are relevant to modulation of signalling via α4ß2 nACh receptors in physiological and pathophysiological conditions.

Structural mechanisms of α7 nicotinic receptor allosteric modulation and activation.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.

Functional α7 nicotinic receptors in human airway smooth muscle increase intracellular calcium concentration and contractility in asthmatics.

Although nicotinic acetylcholine receptors (nAChRs) are commonly associated with neurons in the brain and periphery, recent data indicate that they are also expressed in non-neuronal tissues. We recently found the alpha7 (α7nAChR) subunit is highly expressed in human airway smooth muscle (hASM) with substantial increase in asthmatics, but their functionality remains unknown. We investigated the location and functional role of α7nAChRs in hASM cells from normal versus mild-moderate asthmatic patients. Immunostaining and protein analyses showed α7nAChR in the plasma membrane including in asthmatics. In asthmatic hASM, patch-clamp recordings revealed significantly higher functional homomeric α7nAChR channels. Real-time fluorescence imaging showed nicotine, via α7nAChR, increases intracellular Ca2+ ([Ca2+]i) independent of ACh effects, particularly in asthmatic hASM, while cellular traction force microscopy showed nicotine-induced contractility including in asthmatics. These results indicate functional homomeric and heteromeric nAChRs that are increased in asthmatic hASM, with pharmacology that likely differ owing to different subunit interfaces that form the orthosteric sites. nAChRs may represent a novel target in alleviating airway hyperresponsiveness in asthma.NEW & NOTEWORTHY Cigarette smoking and vaping exacerbate asthma. Understanding the mechanisms of nicotine effects in asthmatic airways is important. This study demonstrates that functional alpha7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in human airway smooth muscle, including from asthmatics, and enhance intracellular calcium and contractility. Although a7nAChRs are associated with neuronal pathways, α7nAChR in smooth muscle suggests inhaled nicotine (e.g., vaping) can directly influence airway contractility. Targeting α7nAChR may represent a novel approach to alleviating airway hyperresponsiveness in asthma.

Impact on AChR open channel noise by pore-peripheral salt bridge depends on voltage and divalent cations.

Mechanisms behind the fluctuations in the ionic current through single acetylcholine receptor (AChR) channels have remained elusive. In a recent study of muscle AChR we showed that mutation of a conserved intramembrane salt bridge in the ß- and δ-subunits markedly increased fluctuations in the open channel current that extended from low to high frequency. Here, we show that extracellular divalent cations reduce the high-frequency fluctuations and increase the low-frequency fluctuations. The low-frequency fluctuations are shown to arise from steps between two current levels, with the ratio of the time at each level changing e-fold for a 70 mV increase in membrane potential, indicating modulation by a charged element within the membrane field. Increasing the charge on the ion selectivity filter biases the ratio of current levels equivalent to a 50 mV increase in membrane potential but does not alter the voltage dependence of the ratio. The magnitudes of the voltage dependence and voltage bias allow estimates of the distance between the ion selectivity filter and the voltage-sensing element. Studies with either calcium or magnesium show that the two divalent cations synergize to increase the low-frequency fluctuations, whereas they act independently to decrease the high-frequency fluctuations, indicating multiple divalent cation binding sites. Molecular dynamics simulations applied to the structure of the Torpedo AChR reveal that mutation of the salt bridge alters the equilibrium positions and dynamics of residues local to the site of the mutation and within the adjacent ion selectivity filter in a calcium-dependent manner. Thus, disruption of a conserved intramembrane salt bridge in the muscle AChR induces fluctuations in open channel current that are sensitive to divalent cation binding at multiple sites and modulated by a charged element within the membrane field.

Genetic Variant in Nicotinic Receptor α4-Subunit Causes Sleep-Related Hyperkinetic Epilepsy via Increased Channel Opening.

We describe genetic and molecular-level functional alterations in the α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) from a patient with sleep-related hyperkinetic epilepsy and a family history of epilepsy. Genetic sequencing revealed a heterozygous variant c.851C>G in the CHRNA4 gene encoding the α4 subunit, resulting in the missense mutation p.Ser284Trp. Patch clamp recordings from genetically engineered nAChRs incorporating the α4-Ser284Trp subunit revealed aberrant channel openings in the absence of agonist and markedly prolonged openings in its presence. Measurements of single channel current amplitude distinguished two pentameric stoichiometries of the variant nAChR containing either two or three copies of the α4-Ser284Trp subunit, each exhibiting aberrant spontaneous and prolonged agonist-elicited channel openings. The α4-Ser284 residue is highly conserved and located within the M2 transmembrane α-helix that lines the ion channel. When mapped onto the receptor's three-dimensional structure, the larger Trp substitution sterically clashes with the M2 α-helix from the neighboring subunit, promoting expansion of the pore and stabilizing the open relative to the closed conformation of the channel. Together, the clinical, genetic, functional, and structural observations demonstrate that α4-Ser284Trp enhances channel opening, predicting increased membrane excitability and a pathogenic seizure phenotype.

Stoichiometry-selective modulation of α4ß2 nicotinic ACh receptors by divalent cations.

BACKGROUND AND PURPOSE: α4ß2 nicotinic ACh receptors (nAChRs) comprise the most abundant class of nAChRs in the nervous system. They assemble in two stoichiometric forms, each exhibiting distinct functional and pharmacological signatures. However, whether one or both forms are modulated by calcium or magnesium has not been established. EXPERIMENTAL APPROACH: To assess the functional consequences of calcium and magnesium, each stoichiometric form was expressed in clonal mammalian fibroblasts and single-channel currents were recorded in the presence of a range of ACh concentrations. KEY RESULTS: In the absence of divalent cations, each stoichiometric form exhibits high unitary conductance and simple gating kinetics composed of solitary channel openings or short bursts of openings. However, in the presence of calcium and magnesium, the conductance and gating kinetics change in a stoichiometry-dependent manner. Calcium and magnesium reduce the conductance of both stoichiometric forms, with each cation producing an equivalent reduction, but the reduction is greater for the (α4)2 (ß2)3 form. Moreover, divalent cations promote efficient channel opening of the (α4)3 (ß2)2 stoichiometry, while minimally affecting the (α4)2 (ß2)3 stoichiometry. For the (α4)3 (ß2)2 stoichiometry, at high but not low ACh concentrations, calcium in synergy with magnesium promote clustering of channel openings into episodes of many openings in quick succession. CONCLUSION AND IMPLICATIONS: Modulation of the α4ß2 nAChR by divalent cations depends on the ACh concentration, the type of cation and the subunit stoichiometry. The functional consequences of modulation are expected to depend on the regional distributions of the stoichiometric forms and synaptic versus extrasynaptic locations of the receptors.

Unmasking coupling between channel gating and ion permeation in the muscle nicotinic receptor.

Whether ion channel gating is independent of ion permeation has been an enduring, unresolved question. Here, applying single channel recording to the archetypal muscle nicotinic receptor, we unmask coupling between channel gating and ion permeation by structural perturbation of a conserved intramembrane salt bridge. A charge-neutralizing mutation suppresses channel gating, reduces unitary current amplitude, and increases fluctuations of the open channel current. Power spectra of the current fluctuations exhibit low- and high-frequency Lorentzian components, which increase in charge-neutralized mutant receptors. After aligning channel openings and closings at the time of transition, the average unitary current exhibits asymmetric relaxations just after channel opening and before channel closing. A theory in which structural motions contribute jointly to channel gating and ion conduction describes both the power spectrum and the current relaxations. Coupling manifests as a transient increase in the open channel current upon channel opening and a decrease upon channel closing.

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.

NACHO and 14-3-3 promote expression of distinct subunit stoichiometries of the α4ß2 acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of pentameric ligand-gated ion channels, and in neuronal tissues, are assembled from various types of α- and ß-subunits. Furthermore, the subunits α4 and ß2 assemble in two predominant stoichiometric forms, (α4)2(ß2)3 and (α4)3(ß2)2, forming receptors with dramatically different sensitivity to agonists and allosteric modulators. However, mechanisms by which the two stoichiometric forms are regulated are not known. Here, using heterologous expression in mammalian cells, single-channel patch-clamp electrophysiology, and calcium imaging, we show that the ER-resident protein NACHO selectively promotes the expression of the (α4)2(ß2)3 stoichiometry, whereas the cytosolic molecular chaperone 14-3-3η selectively promotes the expression of the (α4)3(ß2)2 stoichiometry. Thus, NACHO and 14-3-3η are potential physiological regulators of subunit stoichiometry, and are potential drug targets for re-balancing the stoichiometry in pathological conditions involving α4ß2 nAChRs such as nicotine dependence and epilepsy.

Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.

A novel fast-channel myasthenia caused by mutation in ß subunit of AChR reveals subunit-specific contribution of the intracellular M1-M2 linker to channel gating.

Genetic variants causing the fast-channel congenital myasthenic syndrome (CMS) have been identified in the α, δ, and ε but not the ß subunit of acetylcholine receptor (AChR). A 16-year-old girl with severe myasthenia had low-amplitude and fast-decaying miniature endplate potentials. Mutation analysis revealed two heteroallelic variants in CHRNB1 encoding the AChR ß subunit: a novel c.812C>T (p.P248L) variant in M1-M2 linker (p.P271L in HGVS nomenclature), and a ~430 bp deletion causing loss of exon 8 leading to frame-shift and a premature stop codon (p.G251Dfs*21). P248 is conserved in all ß subunits of different species, but not in other AChR subunits. Measurements of radio-labeled α-bungarotoxin binding show that ßP248L reduces AChR expression to 60% of wild-type. Patch clamp recordings of ACh-elicited single channel currents demonstrate that ßP248L shortens channel opening bursts from 3.3 ms to 1.2 ms, and kinetic analyses predict that the decay of the synaptic response is accelerated 2.4-fold due to reduced probability of channel reopening. Substituting ßP248 with threonine, alanine or glycine reduces the burst duration to 2.3, 1.7, and 1.5 ms, respectively. In non-ß subunits, substituting leucine for residues corresponding to ßP248 prolongs the burst duration to 4.5 ms in the α subunit, shortens it to 2.2 ms in the δ subunit, and has no effect in the ε subunit. Conversely, substituting proline for residues corresponding to ßP248 prolongs the burst duration to 8.7 ms in the α subunit, to 4.6 ms in the δ subunit, but has no effect in the ε subunit. Thus, this fast channel CMS is caused by the dual defects of ßP248L in reducing expression of the mutant receptor and accelerating the decay of the synaptic response. The results also reveal subunit-specific contributions of the M1-M2 linker to the durations of channel opening bursts.

Slow-channel myasthenia due to novel mutation in M2 domain of AChR delta subunit.

OBJECTIVE: To characterize the molecular and phenotypic basis of a severe slow-channel congenital myasthenic syndrome (SCCMS). METHODS: Intracellular and single-channel recordings from patient endplates; alpha-bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure. RESULTS: Among 24 variants reported to cause SCCMS only two appear in the AChR δ-subunit. We here report a 16-year-old patient harboring a novel δL273F mutation (δL294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR δ subunit. Kinetic analyses with ACh and the weak agonist choline indicate that δL273F prolongs the channel opening bursts 9.4-fold due to a 75-fold increase in channel gating efficiency, whereas a previously identified εL269F mutation (εL289F in HGVS nomenclature) at an equivalent location in the AChR ε-subunit prolongs channel opening bursts 4.4-fold due to a 30-fold increase in gating efficiency. Structural modeling of AChR predicts that inter-helical hydrophobic interactions between the mutant residue in the δ and ε subunit and nearby M2 domain residues in neighboring α subunits contribute to structural stability of the open relative to the closed channel states. INTERPRETATION: The greater increase in gating efficiency by δL273F than by εL269F explains why δL273F has more severe clinical effects. Both δL273F and εL269F impair channel gating by disrupting hydrophobic interactions with neighboring α-subunits. Differences in the extent of impairment of channel gating in δ and ε mutant receptors suggest unequal contributions of ε/α and δ/α subunit pairs to gating efficiency.

Structural basis for α-bungarotoxin insensitivity of neuronal nicotinic acetylcholine receptors.

The ten types of nicotinic acetylcholine receptor α-subunits show substantial sequence homology, yet some types confer high affinity for α-bungarotoxin, whereas others confer negligible affinity. Combining sequence alignments with structural data reveals three residues unique to α-toxin-refractory α-subunits that coalesce within the 3D structure of the α4ß2 receptor and are predicted to fit between loops I and II of α-bungarotoxin. Mutating any one of these residues, Lys189, Ile196 or Lys153, to the α-toxin-permissive counterpart fails to confer α-bungarotoxin binding. However, mutating both Lys189 and Ile196 affords α-bungarotoxin binding with an apparent dissociation constant of 104 nM, while combining mutation of Lys153 reduces the dissociation constant to 22 nM. Analogous residue substitutions also confer high affinity α-bungarotoxin binding upon α-toxin-refractory α2 and α3 subunits. α4ß2 receptors engineered to bind α-bungarotoxin exhibit slow rates of α-toxin association and dissociation, and competition by cholinergic ligands typical of muscle nicotinic receptors. Receptors engineered to bind α-bungarotoxin co-sediment with muscle nicotinic receptors on sucrose gradients, and mirror single channel signatures of their α-toxin-refractory counterparts. Thus the inability of α-bungarotoxin to bind to neuronal nicotinic receptors arises from three unique and interdependent residues that coalesce within the receptor's 3D structure.

Potentiation of a neuronal nicotinic receptor via pseudo-agonist site.

Neuronal nicotinic receptors containing α4 and ß2 subunits assemble in two pentameric stoichiometries, (α4)3(ß2)2 and (α4)2(ß2)3, each with distinct pharmacological signatures; (α4)3(ß2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(ß2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(ß2)2 receptors, we show that mutations at either the principal face of the ß2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the ß2-α4 pseudo-agonist sites, the α4-α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the ß2 subunit at either ß2-α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4-α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the ß2-α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(ß2)2 receptor, the findings have implications for structure-guided drug design.

Alcohol reduces muscle fatigue through atomistic interactions with nicotinic receptors.

Alcohol consumption affects many organs and tissues, including skeletal muscle. However, the molecular mechanism of ethanol action on skeletal muscle remains unclear. Here, using molecular dynamics simulations and single channel recordings, we show that ethanol interacts with a negatively charged amino acid within an extracellular region of the neuromuscular nicotinic acetylcholine receptor (nAChR), thereby altering its global conformation and reducing the single channel current amplitude. Charge reversal of the negatively charged amino acid abolishes the nAChR-ethanol interaction. Moreover, using transgenic animals harboring the charge-reversal mutation, ex vivo measurements of muscle force production show that ethanol counters fatigue in wild type but not homozygous αE83K mutant animals. In accord, in vivo studies of motor coordination following ethanol administration reveal an approximately twofold improvement for wild type compared to homozygous mutant animals. Together, the converging results from molecular to animal studies suggest that ethanol counters muscle fatigue through its interaction with neuromuscular nAChRs.

Full and partial agonists evoke distinct structural changes in opening the muscle acetylcholine receptor channel.

The muscle acetylcholine (ACh) receptor transduces a chemical into an electrical signal, but the efficiency of transduction, or efficacy, depends on the particular agonist. It is often presumed that full and partial agonists elicit the same structural changes after occupancy of their binding sites but with differing speed and efficiency. In this study, we tested the alternative hypothesis that full and partial agonists elicit distinct structural changes. To probe structural changes, we substituted cysteines for pairs of residues that are juxtaposed in the three-dimensional structure and recorded agonist-elicited single-channel currents before and after the addition of an oxidizing reagent. The results revealed multiple cysteine pairs for which agonist-elicited channel opening changes after oxidative cross-linking. Moreover, we found that the identity of the agonist determined whether cross-linking affects channel opening. For the αD97C/αY127C pair at the principal face of the subunit, cross-linking markedly suppressed channel opening by full but not partial agonists. Conversely, for the αD97C/αK125C pair, cross-linking impaired channel opening by the weak agonist choline but not other full or partial agonists. For the αT51C/αK125C pair, cross-linking enhanced channel opening by the full agonist ACh but not other full or partial agonists. At the complementary face of the subunit, cross-linking between pairs within the same ß hairpin suppressed channel opening by ACh, whereas cross-linking between pairs from adjacent ß hairpins was without effect for all agonists. In each case, the effects of cross-linking were reversed after addition of a reducing reagent, and receptors with single cysteine substitutions remained unaltered after addition of either oxidizing or reducing reagents. These findings show that, in the course of opening the receptor channel, different agonists elicit distinct structural changes.

Mutations causing congenital myasthenia reveal principal coupling pathway in the acetylcholine receptor ε-subunit.

We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the ß8-ß9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the ß1-ß2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling between conserved residues within the principal coupling pathway of the α-subunit, but also between corresponding residues in the ε-subunit.

Nicotinic acetylcholine receptors at the single-channel level.

Over the past four decades, the patch clamp technique and nicotinic ACh (nACh) receptors have established an enduring partnership. Like all good partnerships, each partner has proven significant in its own right, while their union has spurred innumerable advances in life science research. A member and prototype of the superfamily of pentameric ligand-gated ion channels, the nACh receptor is a chemo-electric transducer, binding ACh released from nerves and rapidly opening its channel to cation flow to elicit cellular excitation. A subject of a Nobel Prize in Physiology or Medicine, the patch clamp technique provides unprecedented resolution of currents through single ion channels in their native cellular environments. Here, focusing on muscle and α7 nACh receptors, we describe the extraordinary contribution of the patch clamp technique towards understanding how they activate in response to neurotransmitter, how subtle structural and mechanistic differences among nACh receptor subtypes translate into significant physiological differences, and how nACh receptors are being exploited as therapeutic drug targets. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc/.