M2-M3 Loop Research Articles

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit β1-β2 and M2-M3 loops, with agonist binding re-positioning a key β1-β2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9’ hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.

The role of zinc binding sites in the cation diffusion facilitator YiiP.

YiiP is a zinc/proton antiporter that couples zinc export to the proton motive force. YiiP binds zinc at three sites: site A in the transmembrane domain, site B in a cytoplasmic loop between transmembrane helices M2 and M3, and site C in the cytoplasmic, C-terminal domain (CTD). Previous Cryo-EM analysis revealed that global removal of zinc by EDTA produced a novel inward occluded state that was postulated to be an intermediate between the inward and outward facing states. In this study we introduced mutations to study binding affinities and conformational effects of each individual site. Cryo-EM analysis showed that disruption of site A produces little change, but that disruption of site B induces the inward occluded state observed previously. Double mutation at site C produced nonnative oligomers, indicating that site C is important for assembly of the native homodimer. Molecular dynamic simulations with zinc removed from site B showed increased mobility of the CTD and disordering of the M2M3 loop in agreement with the structural studies. Binding affinity was quantified by microscale thermophoresis using either citrate or NTA to buffer the concentration of free zinc. The results demonstrate nanomolar affinity at sites A and C and micromolar affinity at site B. Site A is highly sensitive to pH changes with Kd increasing from 1 nM to 302 μM as pH decreases from 7.4 to 5.6. Based on these observations, we propose a transport mechanism whereby zinc is initially recruited at site B and that transfer to site A induces conformational changes leading to transport. The pH dependence of site A is key to energy coupling by ensuring high affinity binding from the cytoplasm and then promoting release to the more acidic periplasmic milieau.

α1 Proline 277 Residues Regulate GABAAR Gating through M2-M3 Loop Interaction in the Interface Region.

Cys-loop receptors are a superfamily of transmembrane, pentameric receptors that play a crucial role in mammalian CNS signaling. Physiological activation of these receptors is typically initiated by neurotransmitter binding to the orthosteric binding site, located at the extracellular domain (ECD), which leads to the opening of the channel pore (gate) at the transmembrane domain (TMD). Whereas considerable knowledge on molecular mechanisms of Cys-loop receptor activation was gathered for the acetylcholine receptor, little is known with this respect about the GABAA receptor (GABAAR), which mediates cellular inhibition. Importantly, several static structures of GABAAR were recently described, paving the way to more in-depth molecular functional studies. Moreover, it has been pointed out that the TMD-ECD interface region plays a crucial role in transduction of conformational changes from the ligand binding site to the channel gate. One of the interface structures implicated in this transduction process is the M2-M3 loop with a highly conserved proline (P277) residue. To address this issue specifically for α1β2γ2L GABAAR, we choose to substitute proline α1P277 with amino acids with different physicochemical features such as electrostatic charge or their ability to change the loop flexibility. To address the functional impact of these mutations, we performed macroscopic and single-channel patch-clamp analyses together with modeling. Our findings revealed that mutation of α1P277 weakly affected agonist binding but was critical for all transitions of GABAAR gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative α1P277 at the interface is strongly involved in regulating the receptor gating.

Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease.

Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC50 value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations.

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1-β2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

Displacement of the Na+/K+ pump’s transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases

Cellular survival requires the ion gradients built by the Na+/K+ pump, an ATPase that alternates between two major conformations (E1 and E2). Here we use state-specific engineered-disulfide cross-linking to demonstrate that transmembrane segment 2 (M2) of the pump's α-subunit moves in directions that are inconsistent with distances observed in existing crystal structures of the Na+/K+ pump in E1 and E2. We characterize this movement with voltage-clamp fluorometry in single-cysteine mutants. Most mutants in the M1-M2 loop produced state-dependent fluorescence changes upon labeling with tetramethylrhodamine-6-maleimide (TMRM), which were due to quenching by multiple endogenous tryptophans. To avoid complications arising from multiple potential quenchers, we analyzed quenching of TMRM conjugated to R977C (in the static M9-M10 loop) by tryptophans introduced, one at a time, in M1-M2. This approach showed that tryptophans introduced in M2 quench TMRM only in E2, with D126W and L130W on the same helix producing the largest fluorescence changes. These observations indicate that M2 moves outward as Na+ is deoccluded from the E1 conformation, a mechanism consistent with cross-linking results and with proposals for other P-type 2 ATPases.

Role of Individual Zinc Binding Sites in the Cation Diffusion Facilitator YIIP

YiiP is a dimeric, zinc/proton bacterial antiporter that belongs to the family of Cation Diffusion Facilitators. YiiP contributes to zinc homeostasis by exporting Zn(II) from the cytosol to the periplasm. YiiP has three Zn(II) binding sites: site a in the membrane domain, site B in an intracellular loop between transmembrane helices M2 and M3, and site C harboring two ions at the dimer interface between the C-terminal domains (CTD) of the homodimer. Here we present high-resolution cryoEM structures of YiiP in complex with a Fab antibody fragment in the presence and absence of Zn(II). Comparison of these structures reveals a dramatic conformational change that affects the accessibility of the transport sites. This conformational change includes bending of M2 and M5 helices, disordering of the M2-M3 loop, and a ∼25° kink between the CTD and the membrane domain. These results are consistent with Molecular Dynamics simulations that show how the M2-M3 loop becomes flexible without the stabilizing effect of Zn(II) bound in site B. To further characterize the role of individual Zn(II) sites in these structural changes, single-site mutations were introduced at each site: D51A at site A, D70A at site B, and D287A at site C. All mutations reduced the stoichiometry of Zn(II) binding, measured by atomic absorption spectroscopy, and abolished Zn(II) transport. Cryo-EM structures of the three mutants were obtained from detergent solubilized YiiP-Fab complexes that were fully loaded with Zn(II). Structures of D51A and D287A mutants revealed conformations comparable to the holo state. In contrast, the D70A mutant produced a structure that resembled the apo state. These findings suggest that binding of Zn(II) to site B induces a conformational change that allows Zn(II) to bind to the transport sites in advance of the transport process.

Cholesterol content in the membrane promotes key lipid-protein interactions in a pentameric serotonin-gated ion channel.

Pentameric ligand-gated ion channels (pLGICs), embedded in the lipid membranes of nerve cells, mediate fast synaptic transmission and are major pharmaceutical targets. Because of their complexity and the limited knowledge of their structure, their working mechanisms have still to be fully unraveled at the molecular level. Over the past few years, evidence that the lipid membrane may modulate the function of membrane proteins, including pLGICs, has emerged. Here, we investigate, by means of molecular dynamics simulations, the behavior of the lipid membrane at the interface with the 5-HT3A receptor (5-HT3AR), a representative pLGIC which is the target of nausea-suppressant drugs, in a nonconductive state. Three lipid compositions are studied, spanning different concentrations of the phospholipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and of cholesterol, hence a range of viscosities. A variety of lipid interactions and persistent binding events to different parts of the receptor are revealed in the investigated models, providing snapshots of the dynamical environment at the membrane-receptor interface. Some of these events result in lipid intercalation within the transmembrane domain, and others reach out to protein key sections for signal transmission and receptor activation, such as the Cys-loop and the M2-M3 loop. In particular, phospholipids, with their long hydrophobic tails, play an important role in these interactions, potentially providing a bridge between these two structures. A higher cholesterol content appears to promote lipid persistent binding to the receptor.

Identification of the Binding Site of Bupropion on Serotonin Type 3A Receptors

Bupropion is an FDA approved medication marketed as an anti-depressant, smoking-cessation, and weight loss drug. Presently, the known effects of bupropion include inhibition of norepinephrine and dopamine reuptake, as well as inhibition of cation-selective Cys-loop ion channels like nicotinic acetylcholine and serotonin type 3 A (5-HT3A) receptors. The present study focuses on bupropion's inhibitory capacity with regard to the 5-HT3A receptor. 5-HT3R is a member of the pentameric ligand-gated ion channel superfamily, which also includes nicotinic acetylcholine, γ-aminobutyric acid type A (GABAA), and glycine receptors. These channels are made up of five homologous subunits around a central ion channel pore. Each subunit is divided into extracellular, transmembrane, and intracellular domain. Dysfunction within this superfamily has been linked to neurological disorders such as anxiety, depression, epilepsy, Alzheimer's, and Parkinson's Disease. Currently, the 5-HT3receptor is targeted clinically by anti-emetics and irritable bowel syndrome treatments, but the receptor could potentially become a target for the treatment of anxiety, psychosis, bipolar disorder, and several other neurological disorders. We engineered amino acids in the α-helical M2 and M3 transmembrane segments near the M2M3 loop that is located at the interface of transmembrane and extracellular domains. We used docking studies and site-directed mutagenesis to analyze potential binding site residues for bupropion. Two-electrode voltage-clamp recordings were used to examine the effect of mutations on bupropion inhibition of these engineered 5-HT3A channels expressed in Xenopus oocytes.

Using a Network of Single Site Specific Mutations and Crosslinking Mass Spectrometry (CXMS) to Refine the Structure and Dynamics of the Human Alpha 1 Glycine Receptor (GLYR)

A network of site-specific single Cys-mutations coupled with CX-MS can be used to elucidate a more refined structure of GlyR and obtain a more definitive understanding of pentameric ligand-gated ion channel (pLGIC) allostery. Each Cys-mutant is introduced into an α1 homomeric Cys null background (C41S/C290A/C345S), or in the same background with F207G/A288G mutation that allows non-desensitizing GlyR activation by ivermectin (IVM). State-dependent crosslinking with methanethiosulfonate benzophenone to a single thiol of purified, vesicle reconstituted GlyR are conducted after enriching the receptor in different allosteric states: resting (no ligand), open (F207G/A288G + IVM), or desensitized (excess glycine). Digested peptides are analyzed via liquid chromatography mass spectrometry to identify sites of intra- and intermolecular crosslinking. Tandem MS of mass-shifted precursor ions further refine these distance constraints. Independent comparative studies targeting different single Cys GlyR (M287C, K116C, K206C) provides evidence of allosteric changes between the three states, as well as direct topological information of unresolved regions, most notably the M3-M4 loop, in other high-resolution structures of pLGICs.

Structure of Heteropentameric GABAA Receptors and Receptor-Anchoring Properties of Gephyrin.

γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.

Proline Residues in the Transmembrane/Extracellular Domain Interface Loops Have Different Behaviors in 5-HT3 and nACh Receptors.

Cys-loop receptors are important drug targets that are involved in signaling in the nervous system. The binding of neurotransmitters in the extracellular region of these receptors triggers an allosteric activation mechanism, the full details of which remain elusive, although structurally flexible loops in the interface between the extracellular region of Cys-loop receptors and the pore-forming transmembrane domain are known to play an important role. Here we explore the roles of three largely conserved Pro residues in two of these loops, the Cys-loop and M2-M3 loop, in 5-HT3A and α7 nACh receptors. The data from natural and noncanonical amino acid mutagenesis suggest that in both proteins a Pro is essential in the Cys-loop, probably because of its enhanced ability to form a cis peptide bond, although other factors are also involved. The important characteristics of Pros in the M2-M3 loop, however, differ in these two receptors: in the 5-HT3 receptor, the Pros can be replaced by some charged amino acids resulting in EC50s similar to those of wild-type receptors, while such substitutions in the nACh receptor ablate function. Ala substitution at one of these Pros also has different effects in the two receptors. Thus, our data show that even highly conserved residues can have distinct behaviors in related Cys-loop receptors.

Role of the Cytoplasmatic M3-M4 Loop for the Homopentameric Assembly of a Chimeric Nicotinic Alpha 7 Receptor

It is long known that the nicotinic acetylcholine receptor α7 (CHRNA7R) expresses weakly as a functional acetylcholine-gated ion channel in many cells. Using the blue native PAGE (BN-PAGE) technique for visualization of oligomers, we have previously shown that singly expressed CHRNA7R subunits assemble in X. laevis oocytes to lower order homooligomers, mostly tetramers; only a small fraction of the synthesized CHRNA7R subunits acquire the fully assembled homopentameric state, in which they can escape the endoplasmic reticulum to be routed to the plasma membrane (Nicke et al, FEBS Lett 575, 52-58. 2004). Here we show that a previously described chimeric receptor (Eiselé et al, Nature 366, 479-483, 1993) comprising the extracellular domain of the CHRNA7R fused to the transmembrane spanning and intracellular domains of the 5HT3A receptor (designated rCHRNA7V2015HT3AR chimera) assembled as efficiently to homopentamers as the 5HT3AR. The large cytoplasmatic M3-M4 loop of the 5HT3AR has previously been identified to represent an oligomerization domain that assembles into stable homopentamers when expressed as a soluble protein without flanking transmembrane helices (Pandhare et al, Sci. Rep. 1-9, 2016). To define the 5HT3AR sequence needed for efficient homopentameric assembly, we systematically varied the sequence encoding the M3-M4 loop of the rCHRNA7V2015HT3AR chimera from 5HT3A to CHRNA7R. We analyzed the resulting chimeras by BN-PAGE, SDS-PAGE and two-electrode voltage-clamp electrophysiology. We found that assembly of functional homopentameric receptors was largely abolished when the CHRNA7R sequence was present into the proximal half of the M3-M4 loop. By a detailed mapping of the first half of the M3-M4 loop we identified a sequence of six amino acids being indispensable for efficient subunit assembly. We will interpret these results on the basis of known 3D structures of ligand-gated ion channels.

Differential State-Dependent Crosslinking of Azi-Cholesterol with Human A1 Glycine Receptor using Mass Spectrometry

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Human a1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol:lipid ratios above the cholesterol activity threshold with aliquots of azi-cholesterol. State-dependent crosslinking studies of receptors primarily in its resting (no glycine), desensitized (10mM glycine) and open (F207A/A288G, 30nM ivermectin) states were performed. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsinized, mass fingerprinted by tandem mass spectrometry (MS), and sites of cholesterol crosslinks in peptides were refined by targeted MS/MS. Differential cholesterol crosslinking patterns between resting, desensitized, and open states were observed, highlighting state-dependent differences in GlyR lipid accessibility. Distinct state-dependent crosslinking patterns indicative of alterations in either the lipid environment and/or channel structure were observed throughout GlyR, most prominently observed in the M4 transmembrane helix, ECD loops and regions nearing the bilayer interface, and the large intracellular M3-M4 loop. Strikingly, crosslinking patterns within the M3-M4 loop offer insight into the generalized structure of this unresolved region of all current pLGIC structural models, by suggesting the crosslinked regions of this intracellular loop are intimately associated or buried within the lipid bilayer. Taken together, crosslinking coupled with tandem MS has the capability to accurately probe and define physiological protein frameworks which can aid in the refinement of allosteric modulation and current structural models.

Electrostatics, proton sensor, and networks governing the gating transition in GLIC, a proton-gated pentameric ion channel

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure-function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson-Boltzmann/Debye-Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pKa close to the experimental pH50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pKa at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the highly conserved electrostatic triad R192-D122-D32 and then to Y197-Y119-K248, both located at the extracellular domain-transmembrane domain interface. The second triad controls a cluster of hydrophobic side chains from the M2-M3 loop that is remodeled during the gating transition. We solved 12 crystal structures of GLIC mutants, 6 of them being trapped in an agonist-bound but nonconductive conformation. Combined with previous data, this reveals two branches of a continuous network originating from E35 that reach, independently, the middle transmembrane region of two adjacent subunits. We conclude that GLIC's gating proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site.

Probing Proline Residues in the Prokaryotic Ligand-Gated Ion Channel, ELIC.

Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate pentameric ligand-gated ion channels (pLGICs) and has proven to be a valuable model for understanding the structure and function of this important protein family. There is nevertheless still a question about whether molecular details can be accurately extrapolated from this protein to those found in eukaryotes. Here we explore the role of proline residues (Pros) in ELIC by creating mutant receptors, expressing them in Xenopus laevis oocytes, and using whole-cell voltage-clamp electrophysiology to monitor channel activity. In contrast to eukaryotic pLGICs, proline-to-alanine (Pro-to-Ala) substitution in ELIC mostly resulted in gain of function, and even altering highly conserved Pro residues in M1 and the M2-M3 loop did not ablate function. These substitutions also mostly resulted in ablation of the modulation by Ca2+ observed in wild-type receptors. Substitution of the Pro in the "Cys loop", however, did result in nonfunctional receptors. Probing this residue with noncanonical amino acids revealed a requirement for a substituted amine at this position, as well as a general preference for Pro analogues with greater intrinsic cis biases. We propose there is likely a cis bond at the apex of the Cys loop in this protein, which is consistent with some, but not all, findings from other pLGICs. Overall, the data show that the roles of proline residues are less critical in ELIC than in other pLGICs, supporting other studies that suggest caution must be applied in using data from this prokaryotic receptor to understand molecular details of eukaryotic pLGIC receptor function.

Structural and Functional Studies Uncover Two Networks Stabilizing the Active Form of GLIC, a Bacterial Proton-Gated Pentameric Ion Channel

The proton-activated pentameric ligand-gated ion channel from Gloeobacter violaceus GLIC can be used as a model system to study the structural and functional properties of its eukaryotic counterparts, which mediate signaling transduction in animal neuron cells by controlling permeation of ions flux gated by neurotransmitter binding. Here we used the Finite Difference Poisson-Boltzmann/Debye-Hückel method to predict the pKas of all the Asp and Glu in GLIC both in the active and resting states. Those residues with a high deviation of predicted pKas in both forms were titrated by Fourier-transform infra-red spectroscopy after reconstitution in lipid bilayers. The results showed that E35 is the main key proton-sensor residue. Examination of the active form structure shows that E35 interacts with T158 from Loop F. Here we verified that breaking this interaction hinders the proton-elicited currents. We next probed the interfacial crevice immediately below E35, shaped by Loop F, Q193 (pre-M1) and the neighboring M2-M3 loop, where a previously unnoticed hydrogen bond network may help maintaining the open channel. We show that breaking this hydrophilic network originating from Q193 favors the ion channel in a nonconductive conformation in the crystal state. Replacing side-chains of residues just below Q193, such as Y197 and I201, with other hydrophobic side-chains blocks the receptor in a locally closed form. Two signal transduction networks are proposed, both originating from the key proton-sensing residue E35, loop F and the pre-M1 region: i) one hydrophilic network extends to the M2-M3 loop from the neighboring subunit and ii) one hydrophobic network interacts with the Cys-loop and the M2-M3 loop of the same subunit. Due to the strong conservation of these loop regions, this model could be generalized to the entire pLGIC family.

GABAA Receptor Coupling Junction and Pore GABRB3 Mutations are Linked to Early-Onset Epileptic Encephalopathy

GABAA receptors are brain inhibitory chloride ion channels. Here we show functional analyses and structural simulations for three de novo missense mutations in the GABAA receptor β3 subunit gene (GABRB3) identified in patients with early-onset epileptic encephalopathy (EOEE) and profound developmental delay. We sought to obtain insights into the molecular mechanisms that might link defects in GABAA receptor biophysics and biogenesis to patients with EOEE. The mutant residues are part of conserved structural domains such as the Cys-loop (L170R) and M2-M3 loop (A305V) that form the GABA binding/channel gating coupling junction and the channel pore (T288N), which are functionally coupled during receptor activation. The mutant coupling junction residues caused rearrangements and formation of new hydrogen bonds in the open state, while the mutant pore residue reshaped the pore cavity. Whereas mutant coupling junction residues uncoupled during activation and caused gain of function, the mutant pore residue favoured low conductance receptors and differential sensitivity to diazepam and loss of function. These data reveal novel molecular mechanisms by which EOEE-linked mutations affect GABAA receptor function.

Un-gating and allosteric modulation of a pentameric ligand-gated ion channel captured by molecular dynamics.

Pentameric ligand-gated ion channels (pLGICs) mediate intercellular communication at synapses through the opening of an ion pore in response to the binding of a neurotransmitter. Despite the increasing availability of high-resolution structures of pLGICs, a detailed understanding of the functional isomerization from closed to open (gating) and back is currently missing. Here, we provide the first atomistic description of the transition from open to closed (un-gating) in the glutamate-gated chloride channel (GluCl) from Caenorhabditis Elegans. Starting with the active-state structure solved in complex with the neurotransmitter L-glutamate and the positive allosteric modulator (PAM) ivermectin, we analyze the spontaneous relaxation of the channel upon removal of ivermectin by explicit solvent/membrane Molecular Dynamics (MD) simulations. The μs-long trajectories support the conclusion that ion-channel deactivation is mediated by two distinct quaternary transitions, i.e. a global receptor twisting followed by the radial expansion (or blooming) of the extracellular domain. At variance with previous models, we show that pore closing is exclusively regulated by the global twisting, which controls the position of the β1-β2 loop relative to the M2-M3 loop at the EC/TM domain interface. Additional simulations with L-glutamate restrained to the crystallographic binding mode and ivermectin removed indicate that the same twisting isomerization is regulated by agonist binding at the orthosteric site. These results provide a structural model for gating in pLGICs and suggest a plausible mechanism for the pharmacological action of PAMs in this neurotransmitter receptor family. The simulated un-gating converges to the X-ray structure of GluCl resting state both globally and locally, demonstrating the predictive character of state-of-art MD simulations.

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin.

The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers. Homopentameric receptors assembled from the Caenorhabditis elegans (C. elegans) GluClα (GLC-1) subunit can inherently respond to IVM but not to glutamate (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies respond to both ligands, independently of each other. Glutamate and IVM bind at the interface between adjacent subunits, far away from each other; glutamate in the extracellular ligand-binding domain, and IVM in the ion-channel pore periphery. To understand the importance of putative intersubunit contacts located outside the glutamate and IVM binding sites, we introduced mutations at intersubunit interfaces, between these two binding-site types. Then, we determined the effect of these mutations on the activation of the heteromeric mutant receptors by glutamate and IVM. Amongst these mutations, we characterized an α-subunit point mutation located close to the putative IVM-binding pocket, in the extracellular end of the first transmembrane helix (M1). This mutation (αF276A) moderately reduced the sensitivity of the heteromeric GluClαF276A/βWT receptor to glutamate, and slightly decreased the receptor subunits’ cooperativity in response to glutamate. In contrast, the αF276A mutation drastically reduced the sensitivity of the receptor to IVM and significantly increased the receptor subunits’ cooperativity in response to IVM. We suggest that this mutation reduces the efficacy of channel gating, and impairs the integrity of the IVM-binding pocket, likely by disrupting important interactions between the tip of M1 and the M2-M3 loop of an adjacent subunit. We hypothesize that this physical contact between M1 and the M2-M3 loop tunes the relative orientation of the ion-channel transmembrane helices M1, M2 and M3 to optimize pore opening. Interestingly, pre-exposure of the GluClαF276A/βWT mutant receptor to subthreshold IVM concentration recovered the receptor sensitivity to glutamate. We infer that IVM likely retained its positive modulation activity by constraining the transmembrane helices in a preopen orientation sensitive to glutamate, with no need for the aforementioned disrupted interactions between M1 and the M2-M3 loop.