Cys-loop Research Articles

Closed-Locked and Apo-Resting State Structures of the Human α7 Nicotinic Receptor: A Computational Study.

Nicotinic acetylcholine receptors, belonging to the Cys-loop superfamily of ligand-gated ion channels (LGICs), are membrane proteins present in neurons and at neuromuscular junctions. They are responsible for signal transmission, and their function is regulated by neurotransmitters, agonists, and antagonists drugs. A detailed knowledge of their conformational transition in response to ligand binding is critical to understanding the basis of ligand-receptor interaction, in view of new pharmacological approaches to control receptor activity. However, the scarcity of experimentally derived structures of human channels makes this perspective extremely challenging. To contribute overcoming this issue, we have recently reported structural models for the open and the desensitized states of the human α7 nicotinic receptor. Here, we provide all-atom structural models of the same receptor in two different nonconductive states. The first structure, built via homology modeling and relaxed with extensive Molecular Dynamics simulations, represents the receptor bound to the natural antagonist α-conotoxin ImI. After comparison with available experimental data and computational models of other eukaryotic LGICs, we deem it consistent with the "closed-locked" state. The second model, obtained with simulations from the spontaneous relaxation of the open, agonist-bound α7 structure after ligand removal, recapitulates the characteristics of the apo-resting state of the receptor. These results add to our previous work on the active and desensitized state conformations, contributing to the structural characterization of the conformational landscape of the human α7 receptor and suggesting benchmarks to discriminate among conformations found in experiments or in simulations of LGICs. In particular key interactions at the interface between the extracellular domain and the transmembrane domain are identified, that could be critical to the α7 receptor function.

A mutational and molecular dynamics study of the cys-loop GABA receptor Hco-UNC-49 from Haemonchus contortus: Agonist recognition in the nematode GABA receptor family

The UNC-49 receptor is a unique nematode γ-aminobutyric acid (GABA)-gated chloride channel that may prove to be a novel target for the development of nematocides. Here we have characterized various charged amino acid residues in and near the agonist binding site of the UNC-49 receptor from the parasitic nematode Haemonchus contorts. Utilizing the Caenorhabditis elegans GluCl crystal structure as a template, a model was generated and various charged residues [D83 (loop D), E131 (loop A), H137 (pre-loop E), R159 (Loop E), E185 (Loop B) and R241 (Loop C)] were investigated based on their location and conservation. These residues may contribute to structure, function, and molecular interactions with agonists. It was found that all residues chosen were important for receptor function to varying degrees. Results of the mutational analysis and molecular simulations suggest that R159 may be interacting with D83 by an ionic interaction that may be crucial for general GABA receptor function. We have used the results from this study as well as knowledge of residues involved in GABA receptor binding to identify sequence patterns that may assist in understanding the function of lesser known GABA receptor subunits from parasitic nematodes.

Structure-Function Relationships of Glycine and GABAA Receptors and Their Interplay With the Scaffolding Protein Gephyrin.

Glycine and γ-aminobutyric acid (GABA) are the major determinants of inhibition in the central nervous system (CNS). These neurotransmitters target glycine and GABAA receptors, respectively, which both belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGICs). Interactions of the neurotransmitters with the cognate receptors result in receptor opening and a subsequent influx of chloride ions, which, in turn, leads to hyperpolarization of the membrane potential, thus counteracting excitatory stimuli. The majority of glycine receptors and a significant fraction of GABAA receptors (GABAARs) are recruited and anchored to the post-synaptic membrane by the central scaffolding protein gephyrin. This ∼93 kDa moonlighting protein is structurally organized into an N-terminal G-domain (GephG) connected to a C-terminal E-domain (GephE) via a long unstructured linker. Both inhibitory neurotransmitter receptors interact via a short peptide motif located in the large cytoplasmic loop located in between transmembrane helices 3 and 4 (TM3-TM4) of the receptors with a universal receptor-binding epitope residing in GephE. Gephyrin engages in nearly identical interactions with the receptors at the N-terminal end of the peptide motif, and receptor-specific interaction toward the C-terminal region of the peptide. In addition to its receptor-anchoring function, gephyrin also interacts with a rather large collection of macromolecules including different cytoskeletal elements, thus acting as central scaffold at inhibitory post-synaptic specializations. Dysfunctions in receptor-mediated or gephyrin-mediated neurotransmission have been identified in various severe neurodevelopmental disorders. Although biochemical, cellular and electrophysiological studies have helped to understand the physiological and pharmacological roles of the receptors, recent high resolution structures of the receptors have strengthened our understanding of the receptors and their gating mechanisms. Besides that, multiple crystal structures of GephE in complex with receptor-derived peptides have shed light into receptor clustering by gephyrin at inhibitory post-synapses. This review will highlight recent biochemical and structural insights into gephyrin and the GlyRs as well as GABAA receptors, which provide a deeper understanding of the molecular machinery mediating inhibitory neurotransmission.

Molecular Characterization of Binding Loop E in the Nematode Cys-Loop GABA Receptor.

Nematodes exhibit a vast array of cys-loop ligand-gated ion channels with unique pharmacologic characteristics. However, many of the structural components that govern the binding of various ligands are unknown. The nematode cys-loop GABA receptor uncoordinated 49 (UNC-49) is an important receptor found at neuromuscular junctions that plays an important role in the sinusoidal movement of worms. The unique pharmacologic features of this receptor suggest that there are structural differences in the agonist binding site when compared with mammalian receptors. In this study, we examined each amino acid in one of the main agonist binding loops (loop E) via the substituted cysteine accessibility method (SCAM) and analyzed the interaction of various residues by molecular dynamic simulations. We found that of the 18 loop E mutants analyzed, H142C, R147C, and S157C had significant changes in GABA EC50 and were accessible to modification by a methanethiosulfonate reagent (MTSET) resulting in a change in I GABA In addition, the residue H142, which is unique to nematode UNC-49 GABA receptors, appears to play a negative role in GABA sensitivity as its mutation to cysteine increased sensitivity to GABA and caused the UNC-49 receptor partial agonist 5-aminovaleric acid (DAVA) to behave as a full agonist. Overall, this study has revealed potential differences in the agonist binding pocket between nematode UNC-49 and mammalian GABA receptors that could be exploited in the design of novel anthelmintics.

Structural titration of receptor ion channel GLIC gating by HS-AFM

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein-protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

Multiple regions in the extracellular domain of the glycine receptor determine receptor activity

Glycine receptors (GlyRs) are Cys-loop receptors that mediate fast synaptic inhibition in the brain stem and spinal cord. They are involved in the generation of motor rhythm, reflex circuit coordination, and sensory signal processing and therefore represent targets for therapeutic interventions. The extracellular domains (ECDs) of Cys-loop receptors typically contain many aromatic amino acids, but only those in the receptor binding pocket have been extensively studied. Here, we show that many Phe residues in the ECD that are not located in the binding pocket are also involved in GlyR function. We examined these Phe residues by creating several GlyR variants, characterizing these variants with the two-electrode voltage clamp technique in Xenopus oocytes, and interpreting changes in receptor parameters by using currently available structural information on the open and closed states of the GlyR. Substitution of six of the eight Phe residues in the ECD with Ala resulted in loss of function or significantly increased the EC50 and also altered the maximal response to the partial GlyR agonist taurine compared with glycine in those receptor variants that were functional. Substitutions with other amino acids, combined with examination of nearby residues that could potentially interact with these Phe residues, suggested interactions that could be important for GlyR function, and possibly similar interactions could contribute to the function of other members of the Cys-loop receptor family. Overall, our results suggest that many ECD regions are important for GlyR function and that these regions could inform the design of therapeutic agents targeting GlyR activity.

Neurotoxic peptides and proteins: biomarkers for screening and identifying ion channel subtypes in healthy/diseased conditions and for drug design

Cys-loop receptors comprising nicotinic acetylcholine receptors (nAChRs), glycine-, ionotropic GABA and serotonin receptors exist as several subtypes differing in localization and role in normal functioning/pathologies. Contrary to antibodies, their identification is more efficient with neurotoxic peptides and proteins from animal venoms, highly selectivity for a particular receptor subtype. Naturally-occurring neurotoxins are used in electrophysiology, while their radioactive/fluorescent derivatives help to measure the receptor subtypes at normal state and diseases. Such measurements are important, for example, for α4β2 nAChRs involved in nicotine addiction, for α7 neuronal nAChRs implicated in β-amyloid peptide binding and for “non-neuronal” α7 nAChR regulating anti-inflammatory response and lung cancer growth. X-ray analysis of the neurotoxin complexes with the receptor models shed light on the binding sites and on possible functional effects of certain disease-associated mutations. Neurotoxins give hints for new drugs or become drugs themselves: like a strong analgesic ω-conotoxin (Prialt) blocking one Ca2+ channel subtype. Several α-conotoxins blocking α9/α10 nAChRs are considered as possible analgesics emphasizing that fundamental research of ligand-receptor interactions is important for solving practical medical tasks. Our recent work on (1) nAChRs and other Cys-loop receptors will be illustrated with novel high-affinity α-conotoxins, (2) snake venom α-neurotoxins (3) human endogenous proteins that have the same three-finger folding as α-neurotoxins and regulate the nAChR activities.

Glycine receptors are involved in hippocampal neuronal damage caused by oxygen-glucose deficiency.

Glycine receptors (GlyRs) belong to the family of ligand-gated cys-loop receptors and effectuate fast inhibitory neurotransmission in central nervous system (CNS). They are involved in numerous physiological processes, such as movement, respiration, and processing of sensory information, as well as in regulation of neuronal excitability in different brain regions. GlyRs play important role in the maintenance of excitatory/inhibitory balance in the hippocampus and participate in the development of various brain pathologies. In the present study, we have examined a surface expression of GlyRs by pyramidal neurons and astrocytes in control and after 30 min of oxygen-glucose deprivation (OGD) in the organotypic culture of hippocampal slices. Our investigation has demonstrated a decrease in GlyR-positive staining associated with pyramidal neurons and relative stability of GlyRs expression at the surface of astrocytes 4 hs after OGD. These data indicate that GlyRs dysfunction may represent a significant additional factor leading to enhanced neuronal damage induced by OGD. Pharmacological modulation of GlyRs is a promising venue of research for the correction of negative consequences of oxygen-glucose deficiency.

Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABAA receptor in complex with GABA.

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABAA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABAA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABAA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric α1β1γ2SEM GABAA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the α1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pathway to structural studies of heteromeric GABAA receptors and a framework for rational design of novel therapeutic agents.

Expression and purification of a functional heteromeric GABAA receptor for structural studies.

The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.

The neural γ2α1β2α1β2 gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges.

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ2α1β2α1β2 gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ2α1β2α1β2 has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ2 and 2 α1 subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models-one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2-M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.

Identification of Novel Functionally Important Aromatic Residue Interactions in the Extracellular Domain of the Glycine Receptor.

The extracellular domains (ECDs) of Cys-loop receptors contain many aromatic amino acids, but only relatively few have been well studied. Here we explore the roles of Tyr and Trp residues in the ECD of the glycine receptor and show that four such residues that have not been previously studied (Y24, Y58, W170, and Y197) contribute significantly to the function of the protein. The residues were studied by creating mutant receptors, characterizing them using two-electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using structural information about the open and closed states of the receptor. Alanine substitution of all these residues ablates function or increases the glycine EC50. There are also a number of changes in the relative maximal responses to taurine, a partial agonist, compared to glycine. Further mutations, in combination with structural information, suggest Y24 contributes to an anion-π interaction with a binding loop D residue, Y58 to an S-π interaction stabilizing the Cys loop, W170 to hydrophobic interactions stabilizing the hydrophobic interior of the subunit, and Y197 to a hydrogen bond linking binding loops B and C. These interactions appear to be broadly conserved in other Cys-loop receptors. Thus, we have identified new regions of the glycine receptor that are important contributors to receptor activation and are likely also to contribute to function in other members of this important protein family.

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.

ALLOSTERIC MODULATION OF THE PENTAMERIC LIGAND‐GATED ION CHANNEL ELIC BY FUNCTIONALLY ACTIVE NANOBODIES

Pentameric ligand‐gated ion channels (pLGICs) or Cys‐loop receptors are a class of ion channels involved in fast synaptic transmission in the central and peripheral nervous systems. Members of this family include the nicotinic acetylcholine receptors, serotonin type‐3 receptors, GABAA/C (gamma‐aminobutyric acid) receptors and glycine receptors. These receptors are the target for a wide variety of therapeutics, including benzodiazepines such as diazepam (Valium), which act as positive allosteric modulators (PAMs) of the GABAA receptor and are prescribed as a sedative, anxiolytic or anti‐epileptic. In this study, we employed the prokaryote ligand‐gated ion channel ELIC as a model to understand the structural mechanism of allosteric modulation. We raised nanobodies, which are antibody fragments derived from camelids and have the potential to trap conformationally transient states, and used them as crystallization chaperones for ELIC. Functional characterization of ELIC nanobodies using electrophysiological techniques demonstrated that these nanobodies are active as channel modulators. We demonstrate that nanobodies can either inhibit or potentiate agonist‐evoked channel responses, suggesting that nanobodies can act as negative (NAMs) or positive allosteric modulators (PAMs), respectively. The X‐ray crystal structure of ELIC in complex with a nanobody reveals that a nanobody binds at an allosteric binding site and near to a binding site previously known to mediate potentiation of ELIC by the benzodiazepine flurazepam. Given the structural conservation of allosteric binding sites between ELIC and human receptors our study reveals common mechanisms for allosteric modulation in this family of channels. Our expanded knowledge of the structural determinants of allosteric modulation opens opportunities for the development of new therapeutics targeting pLGICs in ion channel‐related disorders.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of a 5-HT3-ELIC Chimera Revealing the Sites of Action of Modulators.

Cys-loop receptors are major sites of action for many important therapeutically active compounds, but the sites of action of those that do not act at the orthosteric binding site or at the pore are mostly poorly understood. To help understand these, we here describe a chimeric receptor consisting of the extracellular domain of the 5-HT3A receptor and the transmembrane domain of a prokaryotic homologue, ELIC. Alterations of some residues at the coupling interface are required for function, but the resulting receptor expresses well and responds to 5-HT with a lower EC50 (0.34 μM) than that of the 5-HT3A receptor. Partial agonists and competitive antagonists of the 5-HT3A receptor activate and inhibit the chimera as expected. Examination of a range of receptor modulators, including ethanol, thymol, 5-hydroxyindole, and 5-chloroindole, which can affect the 5-HT3A receptor and ELIC, suggest that these compounds act via the transmembrane domain, except for 5-hydroxyindole, which can compete with 5-HT at the orthosteric binding site. The data throw further light on the importance of coupling interface in Cys-loop receptors and provide a platform for examining the mechanism of action of compounds that act in the extracellular domain of the 5-HT3A receptor and the transmembrane domain of ELIC.

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT3A receptors (5-HT3ARs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT3AR can be exchanged between the homomeric 5-HT3AR and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT3AR affect the affinity of nicotine, a competitive antagonist of 5-HT3ARs, in a cooperative manner. Using mutant cycle analyses between the 5-HT3A triad, loop A residues Asn-101 and Glu-102, β9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

The Pros of nACh and 5-HT3 Receptors

Pentameric ligand gated ion channels (pLGIC) mediate fast synaptic transmission in animals, where they are known as Cys-loop receptors. Prokaryotic homologues of these proteins have been found in bacteria, although these do not possess the eponymous Cys-loop. Each receptor is constituted of five subunits which surround a central pore. A detailed analysis of sequences of all these subunits reveals that only the Pro in the Cys-loop is absolutely conserved; this has led to the proposal (which has not been widely adopted) that these receptors should be renamed ‘Pro-loop’ receptors. In addition to this Pro, there are 3 other Pros that are largely conserved and have been shown to play important but different roles. Here we combine old and new data to compare the characteristics of these Pro residues that allow them to have these distinct roles. Pro has a unique chemistry in proteins because it lacks a backbone hydrogen bond, and has a stronger cis-bias, and greater steric bulk at the amine than other amino acids. Determining which of these properties is essential at which location in Cys-loop receptors can only be accomplished using proline analogues, i.e non-conventional amino acids. These were inserted using nonsense suppression into nACh and 5-HT_3 receptors and receptor characteristics were determined using two electrode voltage clamp in Xenopus oocytes. The data reveal some similarities but also some differences in the roles of Pro at equivalent positions, and identify differences in the specific Pro properties that allow these two related Cys-loop receptors to function.

Highly Pure Soluble Chimeras of the Intracellular Domain of Anionic Pentameric Ligand-Gated Ion Channels

The past few years have seen remarkable advances in structural information gleaned from atomic resolution X-ray crystallographs of important anionic and cationic pentameric ligand-gated ion channels (pLGIC) from the Cys-loop superfamily. The highly conserved extracellular domain (ECD) and transmembrane domain (TMD), and the diverse intracellular domain (ICD) constitute each subunit of eukaryotic pLGICs. The ECD and TMD of different Cys-loop receptors are prime targets for several clinical drugs. These drugs are thus notorious for binding indiscriminately to undesired receptors within the superfamily resulting in a wide range of unwanted side-effects. Intriguingly, the marked variability of ICDs between different subtypes can be exploited to aid in the development of newer drugs with refined specific binding. However, the ICD of any Cys-loop receptor has yet to be fully characterized. The two prominent anion-conducting pLGICs, glycine-α1 (Gly-α1) and γ-amino butyric acid type A-ρ1 (GABAA-ρ1) receptors, of the Cys-loop superfamily are critical in mediating postsynaptic inhibition in the central nervous system. Dysfunction of these pLGICs has been implicated in numerous serious neuropsychiatric and neurological diseases. The goal of our current project was to develop a strategy for protein expression and purification of the ICD of Gly-α1 as well as GABAA-ρ1 receptors. Here, we generated chimeras containing maltose-binding protein (MBP) fused to the N-terminus of the ICD of either Gly-α1 receptor or that of GABAA-ρ1 receptor and separated by a short alanine linker. We expressed these chimeras in E. coli and were able to purify both proteins of interest to homogeneity by employing 3-step purification. Further characterization to determine size using size exclusion chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) revealed the oligomeric state. Experiments are underway to gain detailed structural insights.

Expansion of a Transmembrane Cavity Facilitates Anesthetic Potentiation of a Pentameric Ligand Gated Ion Channel

Ligand-gated ion channels are important molecular targets of general anesthetics, some of which have been in use since the mid 19th century. However, the molecular mechanism of these drug-receptor interactions is poorly understood, creating barriers to the development of new anesthetics. Biochemical and electrophysiological studies have shown that general anesthetics bind to small pockets in Cys-loop receptors, also known as pentameric ligand-gated ion channels. In addition, recent X-ray structures of the Gloeobacter violaceus ligand-gated ion channel show differential binding of the common anesthetic propofol in the presence of sensitizing mutations. In this work, we used molecular dynamics simulations to investigate how anesthetic binding relates to receptor structure. We found a correlation between anesthetic occupancy and binding site expansion. This correlation was removed by a mutation that increased functional sensitivity to anesthetic potentiation. Contraction of the apo binding site from the expanded conformation was associated with a pinching motion of two transmembrane helices, this motion being less pronounced in the mutant than in the wild-type channel. The sensitizing mutation also influenced flexibility of the mutated amino acid side chain and the bound anesthetic. These novel dynamic observations enabled us to refine an allosteric mechanism for bimodal anesthetic modulation in the Cys-loop receptor family, and provided testable hypotheses for further structure-function experiments.

Flexibility of a Transmembrane Helix Underlies Dramatic Reversal of Net Anesthetic Effects in a Pentameric Ligand-Gated Ion Channel

Mechanisms of action for general anesthetics have evolved substantially from purely lipid-based theories to models involving specific protein interfaces, with the capacity to allosterically inhibit as well as potentiate human Cys-loop receptors. However, we still lack a structurally detailed mechanism for these bimodal allosteric effects. Recently, we mapped an allosteric model for anesthetic modulation involving multiple state-dependent binding sites, based on new co-crystal structures of the bacterial homolog GLIC and the general anesthetic propofol. Notably, a point mutation (M205W) in the first transmembrane helix facilitated propofol binding in a membrane-accessible intrasubunit site, and conferred primarily potentiating rather than inhibitory net anesthetic effects. Molecular dynamics simulations correlated the potentiated phenotype with changes in backbone flexibility of the first transmembrane helix. Here, we used mutagenesis and two-electrode voltage-clamp electrophysiology to reveal that helix-perturbing residues at the same transmembrane position lead to potent potentiation even by subclinical concentrations of propofol. Interestingly, these variants were so sensitive to propofol that they exhibited persistent potentiation for up to 40 minutes after exposure to moderate concentrations of propofol, in contrast to an almost instantaneous washout in wild-type channels. The extent of this effect depended on propofol exposure time, suggesting involvement of the membrane as a reservoir for a lipid-accessible binding site. Persistent potentiation also appeared to be independent of an inhibitory effect observed at high acute doses, consistent with the presence of a discrete, water-accessible site for inhibition. The resulting mechanism for general anesthetic modulation could bridge the lipid-based Meyer-Overton theory, postulated more than a century ago, with more complex receptor-based models, informing both our understanding of allostery and the development of new anesthetics.