This is a renewal of ongoing R01 funding to define molecular sites of alcohol action in brain and to link alcohol effects on these receptors with specific behaviors such as consumption, reward/aversion, intoxication and physical dependence. During the current period of funding we were successful in providing molecular and even atomic level of analysis of alcohol sites on pentameric ligand-gated (pLGIC) ion channels, including the first crystallographic structure of alcohols bound within a channel protein. We also succeeded in constructing mutant mice with GABAa receptor subunits which are resistant to alcohol modulation allowing us to link specific GABAa receptors with discrete behavioral actions of alcohol. We are in the middle of the fourth year of this project period and to date have 30 publications with 7 more submitted or in preparation. Although we and others have made considerable progress, the molecular sites of alcohol action in the brain are not completely defined. Emerging evidence from human and rodent genetics, as well as recombinant receptors, indicates that several targets which have received only limited attention may be important sites of alcohol action in brain. We propose to define the role of two groups of ligand-gated ion channels, the glycine-activated chloride channels (GlyR) and GABAaRs formed from ? subunits (? GABAaRs) in actions of alcohol at the molecular, electrophysiological and behavioral levels. A strength of this proposal is the combined use of knock-out (KO) and knock-in (KI) mice allowing us to define the importance of the presence of a subunit (KO) and the importance of direct alcohol action on the protein (KI) for behavioral actions of alcohol. An innovative aspect is use of a new technology (transcription activator- like effector nuclease;TALEN) to be employed by our collaborator, Dr. Gregg Homanics, that markedly reduces the time and cost required for construction of mutant mice. In addition to our behavioral studies, mutant mice will be used by our collaborator, Dr. Neil Harrison for electrophysiological studies of glycinergic function in accumbal regions. Several GlyRs and ? GABAaRs are genetically linked with human alcohol dependence and understanding their role in alcohol actions may provide new targets for pharmacotherapies of alcohol abuse and alcoholism.
Even though alcohol (ethanol) has been consumed for thousands of years, we know remarkably little about the way it produces its effects on the brain. An important advance was the identification of specific proteins (neurotransmitter receptors and ion channels) involved in communication between neurons as a target for ethanol. We will define how ethanol acts on these proteins using novel techniques, ranging from the molecular to the behavioral level, combining mutations in mice with electrophysiological and behavioral studies. The final objective is to define key protein sites that can serve as pharmacological or genetic targets for new therapies to alleviate alcohol addiction.
|Howard, Rebecca J; Trudell, James R; Harris, R Adron (2014) Seeking structural specificity: direct modulation of pentameric ligand-gated ion channels by alcohols and general anesthetics. Pharmacol Rev 66:396-412|
|Borghese, Cecilia M; Hicks, Jessica A; Lapid, Daniel J et al. (2014) GABA(A) receptor transmembrane amino acids are critical for alcohol action: disulfide cross-linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites. J Neurochem 128:363-75|
|Blednov, Yuri A; Benavidez, Jillian M; Black, Mendy et al. (2014) GABAA receptors containing ?1 subunits contribute to in vivo effects of ethanol in mice. PLoS One 9:e85525|
|Robinson, Gizelle; Most, Dana; Ferguson, Laura B et al. (2014) Neuroimmune pathways in alcohol consumption: evidence from behavioral and genetic studies in rodents and humans. Int Rev Neurobiol 118:13-39|
|Trudell, James R; Messing, Robert O; Mayfield, Jody et al. (2014) Alcohol dependence: molecular and behavioral evidence. Trends Pharmacol Sci 35:317-23|
|Heusser, Stephanie A; Howard, Rebecca J; Borghese, Cecilia M et al. (2013) Functional validation of virtual screening for novel agents with general anesthetic action at ligand-gated ion channels. Mol Pharmacol 84:670-8|
|Ozburn, Angela Renee; Harris, R Adron; Blednov, Yuri A (2013) Chronic voluntary alcohol consumption results in tolerance to sedative/hypnotic and hypothermic effects of alcohol in hybrid mice. Pharmacol Biochem Behav 104:33-9|
|Blednov, Y A; Benavidez, J M; Black, M et al. (2013) Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication. Neuropharmacology 67:46-56|
|McCracken, Lindsay M; Blednov, Yuri A; Trudell, James R et al. (2013) Mutation of a zinc-binding residue in the glycine receptor *1 subunit changes ethanol sensitivity in vitro and alcohol consumption in vivo. J Pharmacol Exp Ther 344:489-500|
|Sauguet, Ludovic; Howard, Rebecca J; Malherbe, Laurie et al. (2013) Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel. Nat Commun 4:1697|
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