Our past results strongly indicate that exposure to low level hyperbaric helium-oxygen gas mixtures (atmospheric pressure 12 times normal) represents a direct, mechanistic antagonist of ethanol that can be used as a novel tool to identify ethanol's initial site(s) of behavioral action. The primary goal of the present proposal is to use low level hyperbaric exposure (pressure) to test a new hypothesis regarding ethanol's sites of action. This hypothesis, which is based in part on our recent findings suggesting that pressure antagonism is selective for allosteric modulators, proposes that ethanol acts on allosteric coupling pathways that transduce binding events on ligand-gated ion channels. This allosteric coupling hypothesis views ethanol's sites of action from a different perspective that emphasizes a little explored, poorly defined functional region of the receptor (the allosteric coupling pathway) that does not lend itself to manipulation by classical pharmacological techniques (e.g., agonist or antagonist ligands), but can be tested using pressure. The hypothesis suggests the exciting possibility that there may be common physico-chemical and underlying structural characteristics that define the ethanol sensitive regions of receptor proteins and/or their associated membranes that can be identified by pressure. These characteristics may be unique to a given receptor (e.g., GABA/A) or may extend across the subgroup of ligand-gated ion channels currently purported to show ethanol sensitivity (e.g., GABA/A, NMDA, 5HT/3). The proposed studies represent the first step in testing the allosteric coupling hypothesis.
Specific Aims 1 and 2 are to use pressure with behavioral measures (locomotor, anticonvulsant and anesthetic) and biochemical measures (36/Cl- uptake and radiolabeled ligand binding in mouse brain cerebrocortical vesicles) to test three predictions based on the hypothesis. The predictions focus on the manner in which a drug's primary effect on GABA/A receptor function is mediated (allosteric modulation versus direct agonist action, channel blockade or binding), not on whether the ligand initiates its action via receptor binding. PREDICTION 1: Pressure will antagonize the effects of ethanol and one or more of the ligands that act via allosteric modulation of GABA/A receptor function (benzodiazepines; barbiturates and neuroactive steroids). PREDICTION 2: Pressure will not antagonize drugs that act as direct GABA/A receptor agonists or channel blockers (THIP, muscimol or picrotoxin). PREDICTION 3: Pressure will not antagonize direct binding of [3/H] muscimol, [3/H] flunitrazepam or [35/S] TBPS (i.e., binding under conditions that minimize or eliminate allosteric modulation of binding).
Specific Aim 3 is to develop methods for using pressure to test hypotheses regarding ethanol's sites of action at the molecular level. This will be accomplished by repeating key hyperbaric biochemical studies from Aim 2 in stably transfected cell lines which express recombinant GABA/A receptors. We focused the present proposal on the GABA/A receptor and will extend the investigation to other ligand-gated ethanol sensitive ion channels (e.g., NMDA and 5HT/3) in future proposals. Overall, this work will contribute to our long-term goal which is to identify specific targets at which therapeutically relevant agents can be directed to reduce the social problems, loss of lives and tremendous economic costs resulting from the misuse and abuse of alcohol.
| Wyatt, Letisha R; Finn, Deborah A; Khoja, Sheraz et al. (2014) Contribution of P2X4 receptors to ethanol intake in male C57BL/6 mice. Neurochem Res 39:1127-39 |
| Popova, Maya; Trudell, James; Li, Kaixun et al. (2013) Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors. Purinergic Signal 9:621-32 |
| Perkins, Daya I; Trudell, James R; Asatryan, Liana et al. (2012) Charge and geometry of residues in the loop 2 ? hairpin differentially affect agonist and ethanol sensitivity in glycine receptors. J Pharmacol Exp Ther 341:543-51 |
| Ostrovskaya, Olga; Asatryan, Liana; Wyatt, Letisha et al. (2011) Ethanol is a fast channel inhibitor of P2X4 receptors. J Pharmacol Exp Ther 337:171-9 |
| Popova, Maya; Asatryan, Liana; Ostrovskaya, Olga et al. (2010) A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors. J Neurochem 112:307-17 |
| Perkins, Daya I; Trudell, James R; Crawford, Daniel K et al. (2010) Molecular targets and mechanisms for ethanol action in glycine receptors. Pharmacol Ther 127:53-65 |
| Asatryan, Liana; Popova, Maya; Perkins, Daya et al. (2010) Ivermectin antagonizes ethanol inhibition in purinergic P2X4 receptors. J Pharmacol Exp Ther 334:720-8 |
| Perkins, Daya I; Trudell, James R; Crawford, Daniel K et al. (2009) Loop 2 structure in glycine and GABA(A) receptors plays a key role in determining ethanol sensitivity. J Biol Chem 284:27304-14 |
| Perkins, Daya I; Trudell, James R; Crawford, Daniel K et al. (2008) Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors. J Neurochem 106:1337-49 |
| Crawford, Daniel K; Perkins, Daya I; Trudell, James R et al. (2008) Roles for loop 2 residues of alpha1 glycine receptors in agonist activation. J Biol Chem 283:27698-706 |
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