Prokaryotic pLGIC Research Articles

Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs, there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here, we have characterized the structure and dynamics of DeCLIC through cryoelectron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium-binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid-body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium-binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family.

Cryo-EM and small-angle scattering of a pentameric ligand-gated ion channel reveals a dynamic regulatory domain

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is an architectural diversity involving N-terminal domains (NTDs) not found for the eukaryotic relatives, exemplified by the calcium-sensitive channel DeCLIC. Here, we characterized DeCLIC structure using cryogenic electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In both the presence and absence of calcium, cryo-EM reconstructions were similar to a previously reported calcium-bound x-ray structure.

The Intracellular Domain of Pentameric Ligand-Gated Ion Channels and its Effect on Receptor Conductance in GLIC Chimeras

The pentameric ligand-gated ion channel (pLGIC) superfamily includes the nicotinic acetylcholine (nACh), 5-HT3, ɣ-aminobutyric acid type A (GABAA), and glycine receptor. These channels are made up of five homologous subunits surrounding a central ion channel pore and can be divided into three pentameric domains. The extracellular domain (ECD) and transmembrane domain (TMD) of the channels show high levels of homology and have been well studied and structures have been published. In contrast, the intracellular domain (ICD) shows sequence and length divergence in the different receptors. Mutations in the ICD have been shown to alter receptor conductance. Gloeobacter violaceus LGIC (GLIC) is a prokaryotic pLGIC without a native ICD, which can be expressed and studied in Xenopus oocytes. To elucidate the mechanisms by which the ICD affects the channel pathway and conductance we have created chimeras of GLIC ECD and TMD with ICDs of both anion- and cation-conducting pLGICs (GABAρ1, Glyα1, nAChRα7, 5-HT3A). Functional expression of chimeras was first confirmed by applying low pH (pH 5) pulses to oocytes under two-electrode voltage clamp. We have begun to study the single channel conductance and gating kinetics of chimeric channels and confirmed that the GLIC wildtype has a single-channel conductance of 10pS. For the GLIC-5-HT3A-ICD chimera, we expect to see a decrease in conductance as we observed an increase in a construct of 5-HT3A upon removing the ICD. Amino acid engineering in both the chimeras and eukaryotic parent channels will then be used to further understanding of how the ICDs contribute to ion conducting properties.

Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.

Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1β3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

Pentameric Structure of the Soluble Intracellular Domain of 5-HT3A Receptors

The 115 amino acids spanning intracellular domain (ICD) links the third (M3) and fourth (M4) transmembrane segments within each subunit of the 5-HT3A receptor, a cationic homo-pentameric ligand-gated ion channel (pLGIC). The advent of increasingly sophisticated structural studies has unraveled, in part, the three-dimensional structure of eukaryotic as well as prokaryotic pLGICs at atomic resolution. Intriguingly, within all these structures, the ICD has commonly not been completely resolved. To illustrate the lack of completeness, a recent high-resolution 5-HT3A structure, albeit providing novel structural insights, is deprived of 61 amino acids from its ICD resulting in channel activity differing remarkably from wild-type channels. Prokaryotic pLGICs lack the ICD, and instead contain a very short M3-M4 linker. In eukaryotic pLGICs, the ICD exemplifies the diversity of domain architecture, unlike the other two domains - extracellular and transmembrane - which are highly-conserved. Given the expanding functional implications of ICDs in subunit folding, assembly, targeting, posttranslational modifications, reverse allosteric conductance, and protein-protein interactions, it is of great importance to resolve the structures of full-length ICDs in order to bridge this knowledge gap.To attain this set goal, we produced and purified a soluble chimera containing the ICD of the mouse 5-HT3A receptor. Molecular weight determination by both size exclusion chromatography and dynamic light scattering indicates stable pentameric assembly of the chimera in solution. This surprising finding was further corroborated when the chimera was cross-linked with glutaraldehyde vapors and then resolved either by SDS-PAGE or ‘on-chip electrophoresis’, revealing a ladder of discrete covalently-linked oligomers. The unexpected observation that the ICD alone assembles into stable pentamers in solution and its extreme diversity with regard to both length and amino acid-composition is further suggestive of its orphaned role in receptor oligomerization and potentially the specificity thereof.

Probing Molecular Interactions in Erwinia Ligand-Gated Ion Channel (ELIC)

The Erwinia ligand-gated ion channel (ELIC) is a bacterial pentameric ligand ion channel (pLGIC) originally identified in the enterobacterium Erwinia chrysanthemii. ELIC is closely related to a large class of neurotransmitter-gated ion channels (Cys-loop receptors) that underpin fast synaptic transmission in vertebrates and whose malfunction underlies a range of neurological disorders. As structures of Cys-loop receptors have lagged well behind those from prokaryotic pLGICs, ELIC, whose structure was solved in 2008, has proved to be an attractive model system. Here we explore molecular interactions in ELIC revealed by its structure. Of particular interest are intra-protein cation-π interactions, of which there are potentially six in each ELIC subunit: R48-W66; R99-F95; R105-Y38; R141-F142; R199-W160; and R301-W224. To probe these, the amino acids in each pair were swopped, using site directed mutagenesis, in addition to creating mutants with single amino acid changes to Ala and its pair. ELIC function was then probed following expression in HEKT cells using a membrane potential sensitive dye in a Flexstation. WT ELIC revealed GABA concentration dependent increases in fluorescence with a pEC50 of 2.97 + 0.01 (M, n > 6). The mutations had a range of effects from ablation of responses with all substituted amino acids (e.g. R105-Y38) to wild type responses with all substituted amino acids (e.g. R48-W66). The nature and importance of the interactions formed by these amino acids in ELIC, and whether the data can be extrapolated to better understand vertebrate Cys loop receptors, will be discussed.

A Soluble GABAAρ1 Intracellular Domain Chimera for Structural Studies

The GABAAρ1 pentameric ligand-gated ion channel (pLGIC) is an anion-conducting receptor of the Cys-loop superfamily gated by the neurotransmitter γ-aminobutyric acid (GABA). GABAAρ1 and other anionic pLGICs are the targets of pharmacotherapy including general anesthetics, anti-epileptics (anti-convulsants), sedatives, anxiolytics, and muscle relaxants. In an effort to improve current treatments and develop new drugs, further studies characterizing the pLGICs and more specifically their structural elements that contribute to their function are essential. Each subunit of eukaryotic Cys-loop receptors is comprised of three domains: extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD). Several structural studies including X-ray crystallography of prokaryotic as well as eukaryotic pLGICs have elucidated the three-dimensional structures of both the ECD and TMD. However, the complete picture on the three-dimensional structure of the ICD is not available. This is partly due to the lack of the ICD in prokaryotic pLGICs, such as Gleoeobacter violaceus ligand-gated ion channel (GLIC). Furthermore, in order to obtain higher resolution X-ray crystallographs of mammalian Cys-loop receptors, the ICD is frequently modified. Of the three domains, the ICD of Cys-loop receptors in the animal kingdom has the most diversity in terms of amino acid sequence and length. Therefore, drugs selective for the ICD would be an ideal alternative to current pharmacotherapy ridden with many side effects that result from targeting the highly-conserved extracellular or transmembrane domains.Our project focused on the expression and large-scale purification of GABAAρ1 chimeras in amounts that would allow future structural studies. A soluble chimera containing the ICD was over-expressed in E. coli cells and purified to homogeneity using a two-step purification process. We were successful in producing approximately 3-4 mg of purified protein from a liter of bacterial culture.

Mechanisms of Gating and Modulation in Pentameric Ligand Gated Channels

Structures of pentameric ligand-gated channels (pLGIC) have now reached atomic resolution, with some members of the family also captured in multiple functional states. However, it is widely known that pLGIC gating is extensively modulated by membrane lipids. Using prokaryotic pLGIC homologues and chimeric channels, we probe the allosteric mechanism underlying these effects. Here we show by a combination of EPR spectroscopy, X-ray crystallography, and electrophysiology how lipids modulate function by altering the dynamics of transmembrane helices at the membrane-lipid interface.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.

Divergent Roles for M4 in the Gating of Two Prokaryotic Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels (pLGICs) play a fundamental role in synaptic communication and are the sites of action of numerous allosteric modulators. Recently acquired data suggest that the outermost transmembrane helix, M4, is central to the potentiation of gating observed with some allosteric modulators that interact with the transmembrane domain. Specifically, enhanced interactions between M4 and the adjacent transmembrane helices, M1 and M3, promote coupling between the agonist site and transmembrane gate, while weakened interactions inhibit coupling. Here, we explore in detail the functional role of M4 in the gating of two prokaryotic pLGICs, GLIC and ELIC. Ala mutations along the entire length of M4 typically inhibited channel function in GLIC, with the strongest detrimental effects occurring in the C-terminus along the protein-facing surface. In contrast, Ala mutations in ELIC typically led to enhanced channel function with the strongest potentiating effects occurring closer to the N-terminus. Sequential C-terminal deletions of M4 in GLIC led to the abolishment of channel gating and/or trafficking to the cell surface. In contrast, C-terminal M4 deletion mutants in ELIC still gated open in response to agonist, while deletion of eight or more residues led to fast desensitizing channels with gating kinetics similar to those of alpha7 nAChRs. Our results highlight the importance of M4 in channel function, but suggest M4 in ELIC and GLIC have divergent roles in the gating of each prokaryotic pLGIC.

A Shared Binding Site for Propofol and Thiopental in ELIC

The intravenous general anesthetics propofol and thiopental target pentameric ligand-gated ion channels (pLGIC) and inhibit cation-conducting nAChRs. These drugs also inhibit ELIC, a prokaryotic pLGIC. However, the binding sites for these anesthetics are unknown for either nAChRs or ELIC. Here, using photoaffinity labeling, two-electrode voltage clamp electrophysiology and molecular docking, we identified a functionally relevant binding site for thiopental and propofol in ELIC. Molecular docking identified two binding pockets: an intrasubunit site near M265 of TM3, partially overlapped with the previously identified propofol binding site in GLIC; and an intersubunit site near W220 of TM1, which overlaps with the bromoform binding location identified previously. We generated two mutants, one targeted both predicted binding sites (W220F/W224F/M265C) and another targeted only the intrasubunit site (M265C). Functional measurements on Xenopus oocytes expressing the W220F/W224F/M265C mutant show a significant decrease of anesthetic inhibition, with a five-fold increase in the propofol IC50 and abolishment of thiopental inhibition. Interestingly, the M265C mutation alone could produce the same effect as the W220F/W224F/M265C mutant. Photoaffinity labeling experiments with a light-activated derivative of propofol (aziPm), in conjunction with mass spectrometry, confirmed the binding site at M265 for aziPm. Altogether, the results show that propofol and thiopental bind to a common functionally relevant site. This intrasubunit action site may also be shared by other intravenous anesthetics. Research supported by grants from the NIH.

The Prokaryote Ligand-Gated Ion Channel ELIC Captured in a Pore Blocker-Bound Conformation by the Alzheimer’s Disease Drug Memantine

Pentameric ligand-gated ion channels (pLGIC) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules, including the Alzheimer's drug memantine, block ion conduction at vertebrate pLGICs by plugging the channel pore. We show that memantine has similar potency in ELIC, a prokaryotic pLGIC, when it contains an F16'S pore mutation. X-ray crystal structures, using both memantine and its derivative, Br-memantine, reveal that the ligand is localized at the extracellular entryway of the channel pore, and the pore is in a more closed conformation than wild-type ELIC in both the presence and absence of memantine. However, using voltage clamp fluorometry we observe fluorescence changes in opposite directions during channel activation and pore block, revealing an additional conformational transition not apparent from the crystal structures. These results have important implications for drugs such as memantine, which block channel pores.

Biochemical and Functional Characterization of a Novel Prokaryote Ligand-Gated Ion Channel CLIC

Crystal structures derived from acetylcholine binding proteins, prokaryotic and invertebrate pentameric ligand-gated ion channels (pLGIC) have proven to be valuable tools to study underlying mechanisms concerning ligand binding, channel gating and ion permeation. The aim of this study is to further expand this knowledge by identifying suitable targets for structural studies. Here we report the identification of a novel prokaryotic pLGIC, referred to as CLIC. CLIC is readily expressed in E.coli cells and can be extracted from the cell membrane using detergents in a homogenous and monodisperse state. Purification using a maltose-binding protein affinity tag and size-exclusion chromatography yields milligram quantities of the receptor in a biochemically stable state. CLIC produces crystals within one week but the crystal diffraction quality is limited due to anisotropic diffraction, which prevents structural elucidation. Extensive optimization of crystal growth in the presence of different lipids and detergents has failed to improve crystal diffraction. We will describe the biochemical properties of nanobody- and Fab-based complexes of CLIC to enhance crystal quality. In parallel, we have conducted a ligand screening using CLIC expressed in Xenopus oocytes. We discovered several ligands that activate CLIC and can be used as future tools in crystallization trials. Together, these results open perspectives for the structure determination of novel prokaryote pLGICs.

Mechanisms Underlying the Uncoupling of Binding and Gating in the Nicotinic Receptor and its Prokaryotic Homologs

In PC membranes lacking activating cholesterol and anionic lipids, the nicotinic acetylcholine receptor (nAChR) adopts an uncoupled conformation. The uncoupled nAChR retains agonist-binding, but does not usually undergo conformational transitions to the open or desensitized state. The fact that the uncoupled nAChR does not desensitize in most membranes, despite binding agonist, requires that the activation energy between uncoupled and coupled (resting, open, and desensitized) conformations is insurmountably high. Reconstitution of the nAChR into membranes with relatively thick hydrophobic cores, however, leads to slow (minutes to hours) ligand-induced conformational transition to ultimately the desensitized state. The large energy barrier(s) between uncoupled and coupled conformations is(are) consistent with the current model of uncoupling, which proposes that the transmembrane α-helix, M4, from each subunit becomes solvated by lipid, thus weakening interactions between M4 and the Cys-loop. Weakened interactions may lead to an altered Cys-loop conformation that does not interact effectively with the transmembrane domain to elicit agonist-induced channel gating. In this presentation, we will discuss current experiments aimed at elucidating the mechanisms underlying lipid-dependent uncoupling. In particular, we will focus on structural and mutagenesis data obtained using the model prokaryotic pLGICs, ELIC and GLIC. The data highlight the importance of M4 in pLGIC gating and in dictating the lipid sensitivities of the different prokaryotic pLGICs.

Loose Packing of the Extracellular Domain Core of Pentameric Ligand-Gated Ion Channels is Important for Fast Activation and Desensitization

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission. pLGIC activation is initiated by agonist binding to the orthosteric site in the extracellular domain (ECD), which promotes rapid opening of a membrane-embedded ion-conducting pore. Prolonged agonist exposure leads to pLGIC desensitization, which results in channel closure. Eukaryotic pLGICs open in the sub-millisecond to millisecond timescale, and transition into desensitized closed states in tens of milliseconds. To date, we have limited knowledge of the structural elements that underlie the rapid kinetics of activation and desensitization in pLGICs. We previously showed that eukaryotic pLGICs are characterized by loose packing of the ECD hydrophobic core, due to cavities created at two conserved sites by buried side-chains of either polar residues (threonine and serine in nicotinic receptors, nAChRs) or alanines (in GABA-A receptors), and that increasing the packing of the ECD core at these two sites slowed agonist-induced current activation and desensitization rates (Dellisanti et al., Nat Neurosci 2007; Dellisanti and Czajkowski, J Biol Chem 2011). Interestingly, prokaryotic pLGICs, like proton-activated GLIC, activate and desensitize slowly and have a tightly packed ECD core. Here, we generated loose packing mutants of GLIC by mutating V38 and V109 (aligned to nAChR-α1 T52 and S126) to either alanines or threonine and serine, respectively. We expressed the GLIC mutants in Xenopus oocytes and monitored the effects of these mutations on proton-elicited currents using two-electrode voltage clamp. The loose packing GLIC mutants had 2- to 3-fold faster current activation and desensitization rates compared to wild-type GLIC. The effects were larger when both valines (V38AV109A and V38TV109S mutants) were mutated. Our data suggest loose packing of the ECD core is directly implicated in fast activation and desensitization of eukaryotic pLGICs.

Macroscopic Kinetics of Pentameric Ligand Gated Ion Channels: Comparisons between Two Prokaryotic Channels and One Eukaryotic Channel

Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9±5.1 ms and 1.2±0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3±0.3 s and deactivated even slower with a time constant of 4.6±1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying pLGIC gating transitions.

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels

To understand the molecular mechanism of ion permeation in pentameric ligand-gated ion channels (pLGIC), we solved the structure of an open form of GLIC, a prokaryotic pLGIC, at 2.4 Å. Anomalous diffraction data were used to place bound anions and cations. This reveals ordered water molecules at the level of two rings of hydroxylated residues (named Ser6' and Thr2') that contribute to the ion selectivity filter. Two water pentagons are observed, a self-stabilized ice-like water pentagon and a second wider water pentagon, with one sodium ion between them. Single-channel electrophysiology shows that the side-chain hydroxyl of Ser6' is crucial for ion translocation. Simulations and electrostatics calculations complemented the description of hydration in the pore and suggest that the water pentagons observed in the crystal are important for the ion to cross hydrophobic constriction barriers. Simulations that pull a cation through the pore reveal that residue Ser6' actively contributes to ion translocation by reorienting its side chain when the ion is going through the pore. Generalization of these findings to the pLGIC family is proposed.

Structural Basis for Ion Permeation Mechanism in Pentameric Ligand-Gated Ion Channels

Structural studies of ion permeation in pentameric ligand-gated ion channels (pLGIC) need higher resolution crystal structures. We have solved the structure of an open form of GLIC, a prokaryotic pLGIC, at 2.4 Å. It reveals two clearly resolved water pentagons with one sodium ion between them: a self-stabilized ice-like water pentagon at the level of Ser 6' and a second wider water pentagon at the level of Thr 2’, in continuous exchange with Glu −2'. Computational methods predict that the position of the ice-like “tight” pentagon of water is very sensitive to the position of the hydroxyl group of Ser 6’. Electrophysiology on GLIC wild-type and Ser 6’ mutants, including single-channel recordings, confirm that the side chain hydroxyl of Ser 6’ is crucial for ion translocation. Simulations that pull a cation through the pore suggest that the ion remains hydrated during permeation and that residue 6’ actively contribute to ion translocation by reorienting its side chain when the ion is going through the pore. Generalization of these findings to the pLGIC family is proposed.

Propofol Induced Structural Changes in the TMD of the Prokaryotic pLGIC GLIC

General anesthesia, a routine and indispensable tool in modern surgery still remains a poorly understood phenomenon. Recent studies suggest that general anesthetics (GAs) mediate many of their anesthetic actions by binding to intra- or inter-subunit transmembrane domain (TMD) cavities of pentameric ligand gated ion channels (pLGICs) in the brain. The molecular details of this interaction including location of binding sites, conformational changes induced and residues involved in allosteric transduction remain largely unknown. We are using GLIC, a prokaryotic pLGIC to elucidate mechanisms underlying the action of the commonly used intravenous GA, propofol.We individually introduced cysteines at seven sites in the TMD that frame the intra- (I201C in M1, V241C in M2 and T254C in M3), and inter-subunit (N238C, L240C and E242C in M2) cavities as well as the channel lumen (T243C in M2) in GLIC. Propofol slowed the rate of modification of L240C (inter-subunit) and increased the rate of modification of T254C (intra-subunit) suggesting that the extracellular end of the TMD undergoes propofol-induced structural motions that rearrange these cavities and change the local environment at these sites. An increase in modification rate of T254C makes it unlikely that it faces into the propofol binding site as suggested by a recent crystal structure of GLIC with bound propofol (Nury et al., 2011). Moreover, we found that perturbation of residues in the inter-subunit cavity and not those in the intra-subunit cavity caused propofol to potentiate GLIC currents rather than inhibit. Taken together our results show a significant role of the inter-subunit cavity in propofol modulation of pLGICs and reveal conformational changes associated with propofol's actions.