The overall goal is to identify the neurochemical mechanisms responsible for ethanol and benzodiazepine intoxication, tolerance and dependence. We postulate that the GABA-activated chloride channel is an important site of action for these drugs and the proposed research will focus exclusively on this receptor-channel complex. Of particular importance is the use of molecular biological techniques to define genetic differences in alcohol and benzodiazepine sensitivity at the level of the individual genes coding for the GABA-activated chloride channel complex. The proposal has 4 aims: 1. GABA-activated chloride channels are modulated by endogenous regulators such as calcium, cAMP and protein kinase C. The possibility that these regulators modulate the acute actions of ethanol or benzodiazepines or are responsible for tolerance to these drugs will be studied. 2. The channel complex consists of alpha, beta and perhaps gamma subunits; these distinct subunits have been cloned and sequenced, but it is not known if they confer different ethanol or benzodiazepine sensitivity on the channel. We will answer this question by testing drug sensitivity after expression of the cloned subunits in oocytes and by creation of transgenic mice with over- or under-expression of genes coding for specific subunits. The transgenic mice will allow us to ascribe functional roles to the different subunits. 3. Brain mRNA from Long/Short-Sleep (LS/SS) and Diazepam Sensitive/Resistant (DS/DR) mice and RNA from cloned genes will be expressed in Xenopus oocytes to allow electrophysiological study of channel function. Cloned genes from drug- sensitive mice (LS,DS) will also be injected into embryos of drug- resistant mice (SS,DR) to produce transgenic mice with expression of genes from resistant and sensitive lines. 4. Genetic differences in behavioral sensitivity to ethanol or benzodiazepines may be due to differences in nucleotide sequences of the genes coding for the subunits of the GABA-activated chloride channel or sequences of genes coding for promoter regions. Use of LS/SS and DS/DR mice for gene cloning will identify sequence differences between the lines.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Method to Extend Research in Time (MERIT) Award (R37)
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University of Colorado Denver
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