Modulation of α1ß3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix.

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a ß-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABAA α1ß3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the ß+α- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in ß3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [ß3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1ß3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the ß+α- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to ß3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the ß+α- interface for the anesthetic propofol.

Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity.

Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)-ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs.

An intersubunit electrostatic interaction in the GABAA receptor facilitates its responses to benzodiazepines.

Benzodiazepines are positive allosteric modulators of the GABAA receptor (GABAAR), acting at the α-γ subunit interface to enhance GABAAR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABAAR. The molecular rearrangements in the GABAAR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABAAR, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface that were broken by, or formed after, benzodiazepine binding. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys104 in the α1 subunit forms an electrostatic bond with Asp75 of the γ2 subunit after benzodiazepine binding and that this bond stabilizes the positively modified state of the receptor. Substitution of these two residues to cysteine and subsequent covalent linkage between them increased the receptor's sensitivity to low GABA concentrations and decreased its response to benzodiazepines, producing a GABAAR that resembles a benzodiazepine-bound WT GABAAR. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α1 Lys104 and γ2 Asp75 interaction did not play a role in ethanol or neurosteroid modulation of GABAAR, suggesting that different modulators induce different conformational changes in the receptor. These findings may help explain the additive or synergistic effects of modulators acting at the GABAAR.

Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 µM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model for alcohol/anesthetic interaction with human GLRA1.

Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors.

Alcohols inhibit γ-aminobutyric acid type A ρ1 receptor function. After introducing mutations in several positions of the second transmembrane helix in ρ1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. The 6' mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6'M), increased inhibition (T6'V), and small potentiation (T6'Y and T6'F). The 5' mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3' and 9') or no changes (2' and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6' position. Homology models of ρ1 receptors based on the X-ray structure of GluCl showed that the 2', 5', 6', and 9' residues were easily accessible from the ion pore, with 5' and 6' residues from neighboring subunits facing each other; L3' and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated ρ1 receptors [ρ1(I15'S), ρ1(T6'Y), and ρ1(T6'Y,I15'S)] to further characterize the inhibitory alcohol pocket in the wild-type ρ1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6' position. Replacing the bulky 15' residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.

Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors.

BACKGROUND: Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC. METHODS: We directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes. RESULTS: EtOH potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50 µM picrotoxin to the media, a technique that may have applications in expression of other ion channels. CONCLUSIONS: Our results are consistent with a TM mechanism of EtOH modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species.

Molecular modeling of a tandem two pore domain potassium channel reveals a putative binding site for general anesthetics.

Anesthetics are thought to mediate a portion of their activity via binding to and modulation of potassium channels. In particular, tandem pore potassium channels (K2P) are transmembrane ion channels whose current is modulated by the presence of general anesthetics and whose genetic absence has been shown to confer a level of anesthetic resistance. While the exact molecular structure of all K2P forms remains unknown, significant progress has been made toward understanding their structure and interactions with anesthetics via the methods of molecular modeling, coupled with the recently released higher resolution structures of homologous potassium channels to act as templates. Such models reveal the convergence of amino acid regions that are known to modulate anesthetic activity onto a common three- dimensional cavity that forms a putative anesthetic binding site. The model successfully predicts additional important residues that are also involved in the putative binding site as validated by the results of suggested experimental mutations. Such a model can now be used to further predict other amino acid residues that may be intimately involved in the target-based structure-activity relationships that are necessary for anesthetic binding.

Glycine and GABA(A) ultra-sensitive ethanol receptors as novel tools for alcohol and brain research.

A critical obstacle to developing effective medications to prevent and/or treat alcohol use disorders is the lack of specific knowledge regarding the plethora of molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To identify the role of individual receptor subunits in ethanol-induced behaviors, we developed a novel class of ultra-sensitive ethanol receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as four residues in the extracellular loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABA(A)Rs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two-electrode voltage clamp, demonstrated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABA(A)Rs. Collectively, our studies set the stage for using USER technology in genetically engineered animals as a unique tool to increase understanding of the neurobiological basis of the behavioral effects of ethanol.

P2X4 receptors (P2X4Rs) represent a novel target for the development of drugs to prevent and/or treat alcohol use disorders.

Alcohol use disorders (AUDs) have a staggering socioeconomic impact. Few therapeutic options are available, and they are largely inadequate. These shortcomings highlight the urgent need to develop effective medications to prevent and/or treat AUDs. A critical barrier is the lack of information regarding the molecular target(s) by which ethanol (EtOH) exerts its pharmacological activity. This review highlights findings implicating P2X4 receptors (P2X4Rs) as a target for the development of therapeutics to treat AUDs and discusses the use of ivermectin (IVM) as a potential clinical tool for treatment of AUDs. P2XRs are a family of ligand-gated ion channels (LGICs) activated by extracellular ATP. Of the P2XR subtypes, P2X4Rs are expressed the most abundantly in the CNS. Converging evidence suggests that P2X4Rs are involved in the development and progression of AUDs. First, in vitro studies report that pharmacologically relevant EtOH concentrations can negatively modulate ATP-activated currents. Second, P2X4Rs in the mesocorticolimbic dopamine system are thought to play a role in synaptic plasticity and are located ideally to modulate brain reward systems. Third, alcohol-preferring (P) rats have lower functional expression of the p2rx4 gene than alcohol-non-preferring (NP) rats suggesting an inverse relationship between alcohol intake and P2X4R expression. Similarly, whole brain p2rx4 expression has been shown to relate inversely to innate 24 h alcohol preference across 28 strains of rats. Fourth, mice lacking the p2rx4 gene drink more EtOH than wildtype controls. Fifth, IVM, a positive modulator of P2X4Rs, antagonizes EtOH-mediated inhibition of P2X4Rs in vitro and reduces EtOH intake and preference in vivo. These findings suggest that P2X4Rs contribute to EtOH intake. The present review summarizes recent findings focusing on the P2X4R as a molecular target of EtOH action, its role in EtOH drinking behavior and modulation of its activity by IVM as a potential therapy for AUDs.

Alcohol dependence: molecular and behavioral evidence.

Alcohol dependence is a complex condition with clear genetic factors. Some of the leading candidate genes code for subunits of the inhibitory GABAA and glycine receptors. These and related ion channels are also targets for the acute actions of alcohol, and there is considerable progress in understanding interactions of alcohol with these proteins at the molecular and even atomic levels. X-ray structures of open and closed states of ion channels combined with structural modeling and site-directed mutagenesis have elucidated direct actions of alcohol. Alcohol also alters channel function by translational and post-translational mechanisms, including phosphorylation and protein trafficking. Construction of mutant mice with either deletion of key proteins or introduction of alcohol-resistant channels has further linked specific proteins with discrete behavioral effects of alcohol. A combination of approaches, including genome wide association studies in humans, continues to advance the molecular basis of alcohol action on receptor structure and function.

Synthesis and functional survey of new Tacrine analogs modified with nitroxides or their precursors.

A series of new Tacrine analogs modified with nitroxides or pre-nitroxides on 9-amino group via methylene or piperazine spacers were synthesized; the nitroxide or its precursors were incorporated into the Tacrine scaffold. The new compounds were tested for their hydroxyl radical and peroxyl radical scavenging ability, acetylcholinesterase inhibitor activity and protection against Aß-induced cytotoxicity. Based on these assays, we conclude that Tacrine analogs connected to five and six-membered nitroxides via piperazine spacers (9b, 9b/HCl and 12) exhibited the best activity, providing direction for further development of additional candidates with dual functionality (anti Alzheimer's and antioxidant).

Seeking structural specificity: direct modulation of pentameric ligand-gated ion channels by alcohols and general anesthetics.

Alcohols and other anesthetic agents dramatically alter neurologic function in a wide range of organisms, yet their molecular sites of action remain poorly characterized. Pentameric ligand-gated ion channels, long implicated in important direct effects of alcohol and anesthetic binding, have recently been illuminated in renewed detail thanks to the determination of atomic-resolution structures of several family members from lower organisms. These structures provide valuable models for understanding and developing anesthetic agents and for allosteric modulation in general. This review surveys progress in this field from function to structure and back again, outlining early evidence for relevant modulation of pentameric ligand-gated ion channels and the development of early structural models for ion channel function and modulation. We highlight insights and challenges provided by recent crystal structures and resulting simulations, as well as opportunities for translation of these newly detailed models back to behavior and therapy.

Avermectins differentially affect ethanol intake and receptor function: implications for developing new therapeutics for alcohol use disorders.

Our laboratory is investigating ivermectin (IVM) and other members of the avermectin family as new pharmaco-therapeutics to prevent and/or treat alcohol use disorders (AUDs). Earlier work found that IVM significantly reduced ethanol intake in mice and that this effect likely reflects IVM's ability to modulate ligand-gated ion channels. We hypothesized that structural modifications that enhance IVM's effects on key receptors and/or increase its brain concentration should improve its anti-alcohol efficacy. We tested this hypothesis by comparing the abilities of IVM and two other avermectins, abamectin (ABM) and selamectin (SEL), to reduce ethanol intake in mice, to alter modulation of GABAARs and P2X4Rs expressed in Xenopus oocytes and to increase their ability to penetrate the brain. IVM and ABM significantly reduced ethanol intake and antagonized the inhibitory effects of ethanol on P2X4R function. In contrast, SEL did not affect either measure, despite achieving higher brain concentrations than IVM and ABM. All three potentiated GABAAR function. These findings suggest that chemical structure and effects on receptor function play key roles in the ability of avermectins to reduce ethanol intake and that these factors are more important than brain penetration alone. The direct relationship between the effect of these avermectins on P2X4R function and ethanol intake suggest that the ability to antagonize ethanol-mediated inhibition of P2X4R function may be a good predictor of the potential of an avermectin to reduce ethanol intake and support the use of avermectins as a platform for developing novel drugs to prevent and/or treat AUDs.

GABA(A) receptor transmembrane amino acids are critical for alcohol action: disulfide cross-linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites.

Alcohols and inhaled anesthetics modulate GABA(A) receptor (GABA(A)R) function via putative binding sites within the transmembrane regions. The relative position of the amino acids lining these sites could be either inter- or intra-subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA-mediated currents in wild-type and mutant α1ß2γ2 GABA(A)Rs expressed in Xenopus laevis oocytes. We tested for: (i) inter-subunit cross-linking: a cysteine located in α1TM1 [either α1(Q229C) or α1(L232C)] was paired with a cysteine in different positions of ß2TM2 and TM3; (ii) intra-subunit cross-linking: a cysteine located either in ß2TM1 [ß2(T225C)] or in TM2 [ß2(N265C)] was paired with a cysteine in different locations along ß2TM3. Three inter-subunit cysteine pairs and four intra-subunits cross-linked. In three intra-subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra-subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter- or intra-subunit sites is determined by the specific receptor and ligand.

Endothelial fate mapping in mice with pulmonary hypertension.

BACKGROUND: Pulmonary endothelial injury triggers a reparative program, which in susceptible individuals is characterized by neointima formation, vascular narrowing, and the development of pulmonary arterial hypertension. The neointimal cells in human pathological plexiform lesions frequently coexpress smooth muscle α-actin and the endothelial von Willebrand antigen, creating a question about their cellular lineage of origin. METHODS AND RESULTS: Experimental pulmonary hypertension with neointima formation develops in C57Bl/6 mice subjected to left pneumonectomy followed 1 week later by jugular vein injection of monocrotaline pyrrole (20 µg/µL and 1 µL/g; group P/MCTP). Compared with the group vehicle, by day 35, group P/MCTP developed higher right ventricular systolic pressure (54±5 versus 25±2 mm Hg; P<0.01) and right ventricular hypertrophy (0.58±0.16 versus 0.26±0.05; P<0.01). Transgenic vascular endothelial-cadherin Cre recombinase or Tie-2 Cre mice were intercrossed with mTomato/mGreen fluorescent protein double-fluorescent Cre reporter mice to achieve endothelial genetic lineage marking with membrane-targeted green fluorescent protein. In control mice, few endothelial lineage-marked cells lining the lumen of small pulmonary arteries demonstrate expression of smooth muscle α-actin. Concurrent with the development of pulmonary hypertension, endothelial lineage-marked cells are prominent in the neointima and exhibit expression of smooth muscle α-actin and smooth muscle myosin heavy chain. Human pulmonary arterial hypertension neointimal lesions contain cells that coexpress endothelial CD31 or von Willebrand antigen and smooth muscle α-actin. CONCLUSION: Neointimal cells in pulmonary hypertension include contributions from the endothelial genetic lineage with induced expression of smooth muscle α-actin and smooth muscle myosin heavy chain.

Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol.

The molecular mechanism(s) of action of anesthetic, and especially, intoxicating doses of alcohol (ethanol [EtOH]) have been of interest even before the advent of the Research Society on Alcoholism. Recent physiological, genetic, and biochemical studies have pin-pointed molecular targets for anesthetics and EtOH in the brain as ligand-gated ion channel (LGIC) membrane proteins, especially the pentameric (5 subunit) Cys-loop superfamily of neurotransmitter receptors including nicotinic acetylcholine (nAChRs), GABAA (GABAA Rs), and glycine receptors (GlyRs). The ability to demonstrate molecular and structural elements of these proteins critical for the behavioral effects of these drugs on animals and humans provides convincing evidence for their role in the drugs' actions. Amino acid residues necessary for pharmacologically relevant allosteric modulation of LGIC function by anesthetics and EtOH have been identified in these channel proteins. Site-directed mutagenesis revealed potential allosteric modulatory sites in both the trans-membrane domain (TMD) and extracellular domain (ECD). Potential sites of action and binding have been deduced from homology modeling of other LGICs with structures known from crystallography and cryo-electron microscopy studies. Direct information about ligand binding in the TMD has been obtained by photoaffinity labeling, especially in GABAA Rs. Recent structural information from crystallized procaryotic (ELIC and GLIC) and eukaryotic (GluCl) LGICs allows refinement of the structural models including evaluation of possible sites of EtOH action.

Stabilization of the GluCl ligand-gated ion channel in the presence and absence of ivermectin.

Improving our understanding of the mechanisms and effects of anesthetics is a critically important part of neuroscience. The currently dominant theory is that anesthetics and similar molecules act by binding to Cys-loop receptors in the postsynaptic terminal of nerve cells and potentiate or inhibit their function. Although structures for some of the most important mammalian channels have still not been determined, a number of important results have been derived from work on homologous cationic channels in bacteria. However, partly due to the lack of a nervous system in bacteria, there are a number of questions about how these results relate to higher organisms. The recent determination of a structure of the eukaryotic chloride channel, GluCl, is an important step toward accurate modeling of mammalian channels, because it is more similar in function to human Cys-loop receptors such as GABAAR or GlyR. One potential issue with using GluCl to model other receptors is the presence of the large ligand ivermectin (IVM) positioned between all five subunits. Here, we have performed a series of microsecond molecular simulations to study how the dynamics and structure of GluCl change in the presence versus absence of IVM. When the ligand is removed, subunits move at least 2 Å closer to each other compared to simulations with IVM bound. In addition, the pore radius shrinks to 1.2 Å, all of which appears to support a model where IVM binding between subunits stabilizes an open state, and that the relaxed nonIVM conformations might be suitable for modeling other channels. Interestingly, the presence of IVM also has an effect on the structure of the important loop C located at the neurotransmitter-binding pocket, which might help shed light on its partial agonist behavior.

Zinc-dependent modulation of α2- and α3-glycine receptor subunits by ethanol.

BACKGROUND: Strychnine-sensitive glycine receptors (GlyRs) are expressed throughout the brain and spinal cord and are among the strongly supported protein targets of alcohol. This is based largely on studies of the α1-subunit; however, α2- and α3-GlyR subunits are as or more abundantly expressed than α1-GlyRs in multiple forebrain brain areas considered to be important for alcohol-related behaviors, and uniquely some α3-GlyRs undergo RNA editing. Nanomolar and low micromolar concentrations of zinc ions potentiate GlyR function, and in addition to zinc's effects on glycine-activated currents, we have recently shown that physiological concentrations of zinc also enhance the magnitude of ethanol (EtOH)'s effects on α1-GlyRs. METHODS: Using 2-electrode voltage-clamp electrophysiology in oocytes expressing either α2- or α3-GlyRs, we first tested the hypothesis that the effects of EtOH on α2- and α3-GlyRs would be zinc dependent, as we have previously reported for α1-GlyRs. Next, we constructed an α3P185L-mutant GlyR to test whether RNA-edited and unedited GlyRs contain differences in EtOH sensitivity. Last, we built a homology model of the α3-GlyR subunit. RESULTS: The effects of EtOH (20 to 200 mM) on both subunits were greater in the presence than in the absence of 500 nM added zinc. The α3P185L-mutation that corresponds to RNA editing increased sensitivity to glycine and decreased sensitivity to EtOH. CONCLUSIONS: Our findings provide further evidence that zinc is important for determining the magnitude of EtOH's effects at GlyRs and suggest that by better understanding zinc/EtOH interactions at GlyRs, we may better understand the sites and mechanisms of EtOH action.

Inhibition versus potentiation of ligand-gated ion channels can be altered by a single mutation that moves ligands between intra- and intersubunit sites.

Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), I240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.

Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor.

BACKGROUND: Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. METHODS: The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and ß2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. RESULTS: Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between α and ß subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. CONCLUSION: Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.