The overall hypothesis of this grant is that alcohol and related intoxicant-anesthetics elicit some of their acute and initial sedative effects through an interaction with the GABA/benzodiazepine (GABA/BDZ) receptor chloride (CI-) channel complex. Currently, it is still unknown if this interaction occurs directly, that is, at or near the level of the complex, or indirectly via mechanisms mediated by secondary systems. further, it is hypothesized that genetic differences in ethanol sensitivity are due, at least in part, to differences in the structure/function and modulation of the channel complex. Accordingly, there are two general specific aims. First, to examine and characterize the effects of alcohol on the GABA/BDZ receptor CI- channel complex, with a focus on the role of the benzodiazepine site. To accomplish this goal, the effect of acute alcohol exposure on the function and regulation of the GABA/BDZ receptor in rodent brain membrane vesicles will be determined. The effects of ethanol and various benzodiazepine ligands on the kinetic parameters that determine GABA-mediated CI- conductance (rates of CI- exchange and desensitization) will be measured using quench flow techniques.
The second aim i s to attempt to link in vitro sensitivity (ethanol augmentation of GABA-CI- response) with in vivo sensitivity (sedation) to ethanol. Of particular interest is evaluating the nature and validity of the genetic correlation between ethanol and benzodiazepine sensitivity. A component of this second aim is to evaluate the genetic architecture (single- vs polygenic control) of this alcohol phenotype. To accomplish this goal, genetically defined populations of rodents (selected lines and recombinant inbred strains) will be tested for the ability of various agents to modulate the GABA/BDZ receptor CI- channel complex. The effects of flux equilibrium and rates of desensitization will be assessed in brain membranes from recombinant inbred mouse strains. Moreover, the role of lipid microenvironment, glycosylation and intracellular components on the ability of ethanol to modulate the channel function will be examined using a solubilized and purified receptor complex derived from rodents selectively bred for differences in ethanol sensitivity. It is anticipated that these studies will provide insight into the neural and genetic mechanisms responsible for sensitivity to alcohol. This shall in turn enable us to better understand the mode of inheritance of certain behavioral and neurochemical responses to alcohol. Further, these studies will expand our knowledge of the regulation and function of GABA/BDZ receptor CI-channel system.
