The primary goal is to increase understanding of the initial molecular sites and mechanisms causing ethanol's behavioral effects.
The specific aims address two key questions: 1) Does ethanol act by a common physico-chemical mechanism on different ethanol-sensitive ligand-gated ion channels (LGICs) and 2) do """"""""binding pockets"""""""" play a role in causing ethanol-induced changes in LGIC function. The proposal builds on previous findings demonstrating that increased atmospheric pressure (hyperbaric exposure) is a direct, highly selective ethanol antagonist. The proposed studies will use the unique physico-chemical properties and selectivity of pressure antagonism, in combination with behavioral, recombinant and electrophysiological approaches, to address these questions. The rationale is derived from the logic underlying the use of classical pharmacological antagonists to identify the sites and mechanisms of drug action.
Specific Aim I is to develop hyperbaric 2-electrode voltage clamp techniques for measuring ethanol-pressure interactions on LGIC function in the Xenopus oocyte expression system.
Aim I is not a feasibility study, but will focus on transferring already established hyperbaric oocyte electrophysiology techniques to our laboratory.
Specific Aim 2 tests the hypothesis that ethanol acts by a common, operationally defined as pressure sensitive, mechanism on different ligand-gated ion channels (e.g., GABA-A, GABA-rho, Glycine, n-ACh and NMDA receptors) expressed in Xenopus oocytes.
Aim 2 will be accomplished by testing two interrelated predictions derived from the hypothesis.
Specific Aim 3 will test the hypothesis that pressure antagonizes ethanol via acting on putative ethanol """"""""binding"""""""" pockets in LGICs. The goal is to use a direct ethanol antagonist, pressure, in the classical manner to establish a link between the site of antagonism and the site of ethanol action. This will be accomplished by testing three predictions derived from the hypothesis on LGICs expressed in oocytes and in tadpoles. The results of Aim 2 and 3 studies will provide fundamental new insights into the relationships between ethanol's mechanism(s) of action across different LGICs and, in turn, will aid in the development of molecular models of ethanol's site(s) of action. 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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