Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2–M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2–M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating.     

Contributions of conserved residues at the gating interface of glycine receptors

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.     

Evolutionarily conserved allosteric network in the Cys loop family of ligand-gated ion channels revealed by statistical covariance analyses

The Cys loop family of ligand-gated ion channels mediate fast synaptic transmission for communication between neurons. They are allosteric proteins, in which binding of a neurotransmitter to its binding site in the extracellular amino-terminal domain triggers structural changes in distant transmembrane domains to open a channel for ion flow. Although the locations of binding site and channel gating machinery are well defined, the structural basis of the activation pathway coupling binding and channel opening remains to be determined. In this paper, by analyzing amino acid covariance in a multiple sequence alignment, we have identified an energetically interconnected network in the Cys loop family of ligand-gated ion channels. Statistical coupling and correlated mutational analyses along with clustering revealed a highly coupled cluster. Mapping the positions in the cluster onto a three-dimensional structural model demonstrated that these highly coupled positions form an interconnected network linking experimentally identified binding domains through the coupling region to the gating machinery. In addition, these highly coupled positions are also condensed in the transmembrane domains, which are a recent focus for the sites of action of many allosteric modulators. Thus, our results revealed a genetically interconnected network that potentially plays an important role in the allosteric activation and modulation of the Cys loop family of ligand-gated ion channels.     

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.     

Analysis of Hyperekplexia Mutations Identifies Transmembrane Domain Rearrangements That Mediate Glycine Receptor Activation

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes and are therapeutic targets for a wide range of clinical indications. Elucidating the structural differences between their closed and open states may help in designing improved drugs that bias receptors toward the desired conformational state. We recently showed that two new hyperekplexia mutations, Q226E and V280M, induced spontaneous activity in α1 glycine receptors. Gln-226, located near the top of transmembrane (TM) 1, is closely apposed to Arg-271 at the top of TM2 in the neighboring subunit. Using mutant cycle analysis, we inferred that Q226E induces activation via an enhanced electrostatic attraction to Arg-271. This would tilt the top of TM2 toward TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against Ile-225 at the top of TM1 in the neighboring subunit. We infer that this steric repulsion would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the x-ray atomic structures of closed and open state bacterial pLGICs, we propyose that the model of pLGIC activation as outlined here may be broadly applicable across the eukaryotic pLGIC receptor family.     

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.     

Unravelling the conformational plasticity of the extracellular domain of a prokaryotic nAChR homologue in solution by NMR

Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution.     

Nicotinic receptor interloop proline anchors beta1-beta2 and Cys loops in coupling agonist binding to channel gating

Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which alphaArg 209 of the pre-M1 domain and alphaGlu 45 of the beta1-beta2 loop functionally link the two regions, positioning alphaVal 46 of the beta1-beta2 loop in a cavity formed by alphaPro 272 through alphaSer 269 of the M2-M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, alphaVal 46 and alphaVal 132 of the signature Cys loop couple energetically to alphaPro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that alphaPro 272 functionally couples to flanking Val residues extending from the beta1-beta2 and Cys loops within the ACh binding to channel opening transduction pathway.     

Gating Transition of Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are an important family of membrane proteins and play key roles in physiological processes, including signal transduction at chemical synapses. Here, we study the conformational changes associated with the opening and closing of the channel pore. Based on recent crystal structures of two prokaryotic members of the family in open and closed states, respectively, mixed elastic network models are constructed for the transmembrane domain. To explore the conformational changes in the gating transition, a coarse-grained transition path is computed that smoothly connects the closed and open conformations of the channel. We find that the conformational transition involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition.     

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABA_A/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.     

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel.

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.     

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and (125)I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300-301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery.     

Regulation of a pentameric ligand-gated ion channel by a semiconserved cationic lipid-binding site

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure–function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABA_A receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABA_A receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs.     

Allosteric regulation of pentameric ligand-gated ion channels: An emerging mechanistic perspective

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger—a neurotransmitter—into an ion flux through the postsynaptic membrane. They are oligomeric assemblies that provide prototypical examples of allosterically regulated integral membrane proteins. Here, we present an overview of the most recent advances on the signal transduction mechanism based on the X-ray structures of both prokaryotic and invertebrate eukaryotic pLGICs and on atomistic Molecular Dynamics simulations. The present results suggest that ion gating involves a large structural reorganization of the molecule mediated by two distinct quaternary transitions, a global twisting and the blooming of the extracellular domain, which can be modulated by ligand binding at the topographically distinct orthosteric and allosteric sites. The emerging model of gating is consistent with a wealth of functional studies and will boost the development of novel pharmacological strategies.     

Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.     

Computational study of the transmembrane domain of the acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein whose transmembrane domain (TM-domain) is believed to be responsible for channel gating via a hydrophobic effect. In this work, we perform molecular dynamics and Brownian dynamics simulations to investigate the effect of transmembrane potential on the conformation and water occupancy of TM-domain, and the resulting ion permeation events. The results show that the behavior of the hydrophobic gate is voltage-dependent. Large hyperpolarized membrane potential can change the conformation of TM-domain and water occupancy in this region, which may enable ion conduction. An electrostatic gating mechanism is also proposed from our simulations, which seems to play a role in addition to the well-known hydrophobic effect.     

Mobility of Lower MA-Helices for Ion Conduction through Lateral Portals in 5-HT3A Receptors

The intracellular domain of the serotonin type 3A receptor, a pentameric ligand-gated ion channel, is crucial for regulating conductance. Ion permeation through the extracellular vestibule and the transmembrane channel is well understood, whereas the specific ion conduction pathway through the intracellular domain is less clear. The intracellular domain starts with a short loop after the third transmembrane segment, followed by a short α-helical segment, a large unstructured loop, and finally, the membrane-associated MA-helix that continues into the last transmembrane segment. The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a “closed vestibule,” with their lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. The question remains whether the lateral portals or cytosolic constriction conduct ions upon channel opening. In our study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of segments of the MA-helices most distal to the membrane bilayer. Our results indicate that the proximity and orientation for cysteine pairs at I409C/R410C, in close proximity to the lateral windows, and L402C/L403C, at the cytosolic ends of the MA-helices, are conducive for disulfide bond formation. Although conformational changes associated with gating promote cross-linking for I409C/R410C, which in turn decreases channel currents, cross-linking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely.     

Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels

Although the Torpedo nicotinic acetylcholine receptor (nAChR) reconstituted into phosphatidylcholine (PC) membranes lacking cholesterol and anionic lipids adopts a conformation where agonist binding is uncoupled from channel gating, the underlying mechanism remains to be defined. Here, we examine the mechanism behind lipid-dependent uncoupling by comparing the propensities of two prokaryotic homologs, Gloebacter and Erwinia ligand-gated ion channel (GLIC and ELIC, respectively), to adopt a similar uncoupled conformation. Membrane-reconstituted GLIC and ELIC both exhibit folded structures in the minimal PC membranes that stabilize an uncoupled nAChR. GLIC, with a large number of aromatic interactions at the interface between the outermost transmembrane α-helix, M4, and the adjacent transmembrane α-helices, M1 and M3, retains the ability to flux cations in this uncoupling PC membrane environment. In contrast, ELIC, with a level of aromatic interactions intermediate between that of the nAChR and GLIC, does not undergo agonist-induced channel gating, although it does not exhibit the expected biophysical characteristics of the uncoupled state. Engineering new aromatic interactions at the M4-M1/M3 interface to promote effective M4 interactions with M1/M3, however, increases the stability of the transmembrane domain to restore channel function. Our data provide direct evidence that M4 interactions with M1/M3 are modulated during lipid sensing. Aromatic residues strengthen M4 interactions with M1/M3 to reduce the sensitivities of pentameric ligand-gated ion channels to their surrounding membrane environment.     

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the dM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the dM3 transmembrane domain were characterized by two-electrode voltage clamp and ¹²⁵I-labeled a-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300-301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid–exposed transmembrane domain and the nAChR gating machinery.     

Gating mechanisms in Cys-loop receptors

The Cys-loop receptor superfamily of ligand-gated ion channels has a prominent role in neuronal signalling. These receptors are pentamers, each subunit containing ten β-strands in the extracellular domain and four α-helical transmembrane domains (M1–M4). The M2 domain of each subunit lines the intrinsic ion channel pore and residues within the extracellular domain form ligand binding sites. Ligand binding initiates a conformational change that opens the ion-selective pore. The coupling between ligand binding in the extracellular domain and opening of the intrinsic ion channel pore located in the membrane is not fully understood. Several loop structures, such as loop 2, the Cys-loop, the pre-M1 region and the M2–M3 loop have been implicated in receptor activation. The current “conformational change wave” hypothesis suggests that binding of a ligand initiates a rotation of the β-sheets around an axis that passes through the Cys-loop. Due to this rotation, the Cys-loop and loop 2 are displaced. Movement of the M2–M3 loop then twists the M2 domain leading to a separation of the helices and opening of the pore. The publication of a crystal structure of an acetylcholine binding protein and the refined structure of the Torpedo marmorata acetylcholine receptor have improved the understanding of the mechanisms and structures involved in coupling ligand binding to channel gating. In this review, the most recent findings on some of these loop structures will be reported and discussed in view of their role in the gating mechanism.