Properties of Specific Alcohol Binding Sites

Summary

Principal Investigator: James Trudell
Affiliation: Stanford University
Country: USA
Abstract: Alcohol abuse and alcoholism are major health problems. It is likely that a solution to these problems will require an understanding of the effects of alcohol on neuronal ion channel proteins. Specific binding sites for alcohols have been described in the transmembrane (TM) domain of the superfamily of ligand-gated ion channels (LGIC's) that includes glycine (GlyRa1), GABA, nicotinic acetylcholine, and 5-HT3 receptors. Our global hypothesis is that alcohols bind within cavities that are bounded by TM segments of these receptors and preferentially stabilize specific channel substates. Our goal is to define the properties of those sites that mediate binding and efficacy of alcohol and alcohol antagonists in GlyRa1. These binding sites may provide a common motif for binding of alcohols within other classes of ion channels and other important proteins. We will build computational models of binding sites in GlyRa1 and design specific site-directed mutations to test these hypothesis. These mutations will be constructed and tested by our collaborator, Dr. R. A. Harris. Aim 1. We will test the hypothesis that amino acid residues from all four transmembrane helices of GlyRa1 contribute to a binding site for alcohols. We will develop computational models to delineate the dimensions of these sites. We will use these models to predict where covalent binding of alkyl methanethiosulfonate (MTS) reagents would mimic alcohol binding. The predictions will be tested in the Harris laboratory by expressing GlyRa1 containing site-directed cysteine substitutions in oocytes. They will apply MTS reagents to the oocytes and measure the effects on glycine-induced currents. Aim 2. We will test the hypothesis that double site directed cysteine mutations can clarify the refined tertiary structure of the GlyRa1 and distinguish our model from one based on the torpedo nAChR. While the overall structure of our GlyRa1 model and the nAChR model of Unwin are in global agreement, there are important differences in the orientation of transmembrane helices and residues bounding a possible alcohol- binding site. The Harris laboratory will test these predictions by crosslinking site-directed di-cysteines. In summary, knowledge of alcohol binding sites in GlyRa1 will increase our understanding of alcohol action in LGIC's. The results may reveal general motifs for action in other systems affected by alcohol and aid in the design of alcohol antagonists.
Funding Period: 2001-12-01 - 2010-06-30
more information: NIH RePORT

Top Publications

  1. pmc Seeking structural specificity: direct modulation of pentameric ligand-gated ion channels by alcohols and general anesthetics
    Rebecca J Howard
    Department of Chemistry, Skidmore College, Saratoga Springs, NY 12866
    Pharmacol Rev 66:396-412. 2014
  2. pmc Ethanol's molecular targets
    R Adron Harris
    Section of Neurobiology and Waggoner Center for Alcohol and Addiction Research, Institutes for Neuroscience and Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA
    Sci Signal 1:re7. 2008
  3. pmc Normal mode gating motions of a ligand-gated ion channel persist in a fully hydrated lipid bilayer model
    Edward J Bertaccini
    Department of Anesthesia, Stanford University School of Medicine and Beckman Center for Molecular and Genetic Medicine, Stanford, California, USA
    ACS Chem Neurosci 1:552-8. 2010
  4. pmc Molecular mechanism for the dual alcohol modulation of Cys-loop receptors
    Samuel Murail
    Science for Life Laboratory, KTH Royal Institute of Technology and Stockholm University, Stockholm, Sweden
    PLoS Comput Biol 8:e1002710. 2012
  5. pmc Mutation of a zinc-binding residue in the glycine receptor α1 subunit changes ethanol sensitivity in vitro and alcohol consumption in vivo
    Lindsay M McCracken
    Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas, USA
    J Pharmacol Exp Ther 344:489-500. 2013
  6. pmc Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor
    Edward J Bertaccini
    Professor, Department of Anesthesia, Stanford University School of Medicine, Stanford, California, and Anesthesiologist and Intensivist, Palo Alto VA Health Care System, Palo Alto, California Doctoral Candidate, Professor of Computational Structural Biology, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Professor of Chemistry in Anesthesia, Department of Anesthesia, Stanford University School of Medicine
    Anesthesiology 119:1087-95. 2013
  7. pmc Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors
    MAYA POPOVA
    Alcohol and Brain Research Laboratories, Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA, 90089, USA
    Purinergic Signal 9:621-32. 2013
  8. pmc Inhibition versus potentiation of ligand-gated ion channels can be altered by a single mutation that moves ligands between intra- and intersubunit sites
    Torben Brömstrup
    Science for Life Laboratory, KTH Royal Institute of Technology and Stockholm University, 17121 Solna, Sweden
    Structure 21:1307-16. 2013
  9. ncbi Zinc-dependent modulation of α2- and α3-glycine receptor subunits by ethanol
    Lindsay M McCracken
    The Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas
    Alcohol Clin Exp Res 37:2002-10. 2013
  10. pmc Modeling anesthetic binding sites within the glycine alpha one receptor based on prokaryotic ion channel templates: the problem with TM4
    Edward J Bertaccini
    Department of Anesthesia, Stanford University School of Medicine and Beckman Center for Molecular and Genetic Medicine, Stanford, California, United States
    J Chem Inf Model 50:2248-55. 2010

Scientific Experts

  • Liana Asatryan
  • James Trudell
  • Edward J Bertaccini
  • R Adron Harris
  • Rebecca J Howard
  • Daya I Perkins
  • Erik Lindahl
  • Daryl L Davies
  • Daniel K Crawford
  • Ronald L Alkana
  • Samuel Murail
  • Lindsay M McCracken
  • Ingrid A Lobo
  • Mandy L McCracken
  • Torben Brömstrup
  • MAYA POPOVA
  • Minfu Xu
  • Björn Wallner
  • Jelena Todorovic
  • Kaixun Li
  • S John Mihic
  • Mitesh Sanghvi
  • Albert Won
  • Jillian M Benavidez
  • Erik R Lindahl
  • DARYL DAVIES
  • Heinrich Betz
  • Yuri A Blednov
  • RONALD ALKANA
  • Ozge Yoluk
  • Torben Broemstrup
  • C Thetford Smothers
  • John J Woodward
  • Kathryn E Ondricek
  • Pierre Jean Corringer
  • Paul A Slesinger
  • Joydip Das
  • Edward Bertaccini
  • BRIAN T WELSH
  • Cecilia M Borghese
  • Michael P Blanton
  • Ayman K Hamouda
  • Titia Sixma
  • Hugo R Arias
  • Krzysztof Jozwiak
  • Irene Oh
  • Edmond I Eger
  • James M Sonner
  • Michael J Laster
  • Robert Brosnan
  • Mark Liao
  • Jessica Shapiro
  • Douglas L Brutlag

Detail Information

Publications31

  1. pmc Seeking structural specificity: direct modulation of pentameric ligand-gated ion channels by alcohols and general anesthetics
    Rebecca J Howard
    Department of Chemistry, Skidmore College, Saratoga Springs, NY 12866
    Pharmacol Rev 66:396-412. 2014
    ..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. ..
  2. pmc Ethanol's molecular targets
    R Adron Harris
    Section of Neurobiology and Waggoner Center for Alcohol and Addiction Research, Institutes for Neuroscience and Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA
    Sci Signal 1:re7. 2008
    ..In this Review, we describe criteria that should be considered when identifying alcohol binding sites and highlight a number of proteins for which there exists considerable molecular-level evidence for distinct ethanol binding sites...
  3. pmc Normal mode gating motions of a ligand-gated ion channel persist in a fully hydrated lipid bilayer model
    Edward J Bertaccini
    Department of Anesthesia, Stanford University School of Medicine and Beckman Center for Molecular and Genetic Medicine, Stanford, California, USA
    ACS Chem Neurosci 1:552-8. 2010
    ..While these perturbations tend to influence the overall protein motion, this work provides continued support for the iris-like motion model that characterizes gating within the family of ligand-gated ion channels...
  4. pmc Molecular mechanism for the dual alcohol modulation of Cys-loop receptors
    Samuel Murail
    Science for Life Laboratory, KTH Royal Institute of Technology and Stockholm University, Stockholm, Sweden
    PLoS Comput Biol 8:e1002710. 2012
    ..These results demonstrate dramatic effects of the F(14')A mutation on the distribution of ligands, and are consistent with a two-site model of pLGIC inhibition and potentiation...
  5. pmc Mutation of a zinc-binding residue in the glycine receptor α1 subunit changes ethanol sensitivity in vitro and alcohol consumption in vivo
    Lindsay M McCracken
    Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas, USA
    J Pharmacol Exp Ther 344:489-500. 2013
    ....
  6. pmc Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor
    Edward J Bertaccini
    Professor, Department of Anesthesia, Stanford University School of Medicine, Stanford, California, and Anesthesiologist and Intensivist, Palo Alto VA Health Care System, Palo Alto, California Doctoral Candidate, Professor of Computational Structural Biology, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Professor of Chemistry in Anesthesia, Department of Anesthesia, Stanford University School of Medicine
    Anesthesiology 119:1087-95. 2013
    ..Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling...
  7. pmc Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors
    MAYA POPOVA
    Alcohol and Brain Research Laboratories, Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA, 90089, USA
    Purinergic Signal 9:621-32. 2013
    ....
  8. pmc Inhibition versus potentiation of ligand-gated ion channels can be altered by a single mutation that moves ligands between intra- and intersubunit sites
    Torben Brömstrup
    Science for Life Laboratory, KTH Royal Institute of Technology and Stockholm University, 17121 Solna, Sweden
    Structure 21:1307-16. 2013
    ..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. ..
  9. ncbi Zinc-dependent modulation of α2- and α3-glycine receptor subunits by ethanol
    Lindsay M McCracken
    The Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas
    Alcohol Clin Exp Res 37:2002-10. 2013
    ....
  10. pmc Modeling anesthetic binding sites within the glycine alpha one receptor based on prokaryotic ion channel templates: the problem with TM4
    Edward J Bertaccini
    Department of Anesthesia, Stanford University School of Medicine and Beckman Center for Molecular and Genetic Medicine, Stanford, California, United States
    J Chem Inf Model 50:2248-55. 2010
    ..While this model can account for a large proportion of the physicochemical data regarding such proteins, it cannot readily account for the alterations on anesthetic effects that are due to mutations within TM4...
  11. pmc Microsecond simulations indicate that ethanol binds between subunits and could stabilize an open-state model of a glycine receptor
    Samuel Murail
    Department of Theoretical Physics, Royal Institute of Technology, Stockholm, Sweden
    Biophys J 100:1642-50. 2011
    ..Finally, ethanol appears to stabilize the GlyR model built on a presumably open form of the ligand-gated channel. This stabilization could help explain the effects of allosteric ligand binding in Cys-loop receptors...
  12. pmc Alcohol-binding sites in distinct brain proteins: the quest for atomic level resolution
    Rebecca J Howard
    Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Texas 77812, USA
    Alcohol Clin Exp Res 35:1561-73. 2011
    ....
  13. pmc Structural basis for alcohol modulation of a pentameric ligand-gated ion channel
    Rebecca J Howard
    Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, Austin, TX 78712, USA
    Proc Natl Acad Sci U S A 108:12149-54. 2011
    ..These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member...
  14. pmc Ethanol inhibition of constitutively open N-methyl-D-aspartate receptors
    Minfu Xu
    Department of Neurosciences, Medical University of South Carolina, Charleston, SC 29425, USA
    J Pharmacol Exp Ther 340:218-26. 2012
    ..Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits...
  15. pmc Charge and geometry of residues in the loop 2 β hairpin differentially affect agonist and ethanol sensitivity in glycine receptors
    Daya I Perkins
    Alcohol and Brain Research Laboratories, Departments of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA
    J Pharmacol Exp Ther 341:543-51. 2012
    ..This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs...
  16. pmc A transmembrane amino acid in the GABAA receptor β2 subunit critical for the actions of alcohols and anesthetics
    Mandy L McCracken
    Waggoner Center for Alcohol and Addiction Research, University of Texas, Austin, Texas 78712, USA
    J Pharmacol Exp Ther 335:600-6. 2010
    ..Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site...
  17. pmc Ivermectin antagonizes ethanol inhibition in purinergic P2X4 receptors
    Liana Asatryan
    Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA
    J Pharmacol Exp Ther 334:720-8. 2010
    ..Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders...
  18. pmc Molecular targets and mechanisms for ethanol action in glycine receptors
    Daya I Perkins
    Alcohol and Brain Research Laboratories, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA
    Pharmacol Ther 127:53-65. 2010
    ..Recent molecular models of the sites of ethanol action in GlyRs and their implications to our understanding of possible mechanism of ethanol action and novel targets for drug development in GlyRs are discussed...
  19. pmc Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation
    Jelena Todorovic
    Division of Pharmacology and Toxicology, Section of Neurobiology, Waggoner Center for Alcohol and Addiction Research and Institutes for Neuroscience and Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
    Proc Natl Acad Sci U S A 107:7987-92. 2010
    ..This intersubunit electrostatic interaction among GlyR subunits thus contributes to the stabilization of the closed channel state, and its disruption represents a critical step in GlyR activation...
  20. ncbi Accessibility to residues in transmembrane segment four of the glycine receptor
    Ingrid A Lobo
    Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway, MBB 1 124, A4800 Austin, TX 78712, USA
    Neuropharmacology 50:174-81. 2006
    ..Potentiation by isoflurane was significantly reduced for I409C after reaction. These data demonstrate water-accessibility of specific TM4 positions in the GlyR and suggest involvement of these residues with alcohol and anesthetic action...
  21. ncbi The minimum alveolar anesthetic concentration of 2-, 3-, and 4-alcohols and ketones in rats: relevance to anesthetic mechanisms
    Albert Won
    Department of Anesthesia and Perioperative Care, University of California, San Francisco, California 94143 0464, USA
    Anesth Analg 102:1419-26. 2006
    ..The oil/gas partition coefficient predicted potency as well as, or better than, either chain length or oxygen placement. Hydrophilicity, as indicated by the saline/gas partition coefficient, also seemed to influence potency...
  22. ncbi Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of alpha1 glycine receptors
    Daniel K Crawford
    Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089, USA
    J Neurochem 102:2097-109. 2007
    ....
  23. pmc Normal-mode analysis of the glycine alpha1 receptor by three separate methods
    Edward J Bertaccini
    Department of Anesthesia, Stanford University School of Medicine and Beckman Center for Molecular and Genetic Medicine, Stanford, California 94305 5117, USA
    J Chem Inf Model 47:1572-9. 2007
    ..Such analyses may someday allow the use of protein dynamics elucidated via normal-mode calculations as additional endpoints for future drug design...
  24. pmc Cross-linking of sites involved with alcohol action between transmembrane segments 1 and 3 of the glycine receptor following activation
    Ingrid A Lobo
    Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712 1065, USA
    J Neurochem 104:1649-62. 2008
    ....
  25. ncbi Molecular modeling and mutagenesis reveals a tetradentate binding site for Zn2+ in GABA(A) alphabeta receptors and provides a structural basis for the modulating effect of the gamma subunit
    James R Trudell
    Department of Anesthesia, Stanford University School of Medicine, Stanford, California 94305 5117, USA
    J Chem Inf Model 48:344-9. 2008
    ..Sensitivity to Zn2+ was restored in the double mutant receptor, alpha1beta2gamma2 (I282H; K285E), in which the competition with lysine was removed and a more favorable Zn2+ binding site was formed...
  26. ncbi Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein
    Edward J Bertaccini
    Department of Anesthesia, Stanford University School of Medicine and Beckman Center for Molecular and Genetic Medicine, Stanford, California 94305 5117, USA
    J Chem Inf Model 48:855-60. 2008
    ..The results suggest that alterations in receptor dynamics could be a general feature of ligand binding...
  27. ncbi Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors
    Daya I Perkins
    Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, USA
    J Neurochem 106:1337-49. 2008
    ..These findings should help in the development of pharmacological agents that antagonize ethanol...
  28. pmc Roles for loop 2 residues of alpha1 glycine receptors in agonist activation
    Daniel K Crawford
    Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089, USA
    J Biol Chem 283:27698-706. 2008
    ....
  29. doi Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies
    Mitesh Sanghvi
    Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
    Biochim Biophys Acta 1778:2690-9. 2008
    ..Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel...
  30. pmc Loop 2 structure in glycine and GABA(A) receptors plays a key role in determining ethanol sensitivity
    Daya I Perkins
    Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089, USA
    J Biol Chem 284:27304-14. 2009
    ..In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure...
  31. ncbi Homology modeling of a human glycine alpha 1 receptor reveals a plausible anesthetic binding site
    Edward J Bertaccini
    Department of Anesthesia, Stanford University School of Medicine, Stanford, California 94305 5117, USA
    J Chem Inf Model 45:128-35. 2005
    ..This suggests that the binding sites for volatile anesthetics in the LGICs are the cavities formed within the core of transmembrane four-helix bundles...