MODULATORY SITES ON GABA A RECEPTORS

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GABA-A receptors are modulated by many intravenous general anesthetics (IV GAs), at clinically relevant doses. Low concentrations of IV GAs, such as the anesthetic steroids, barbiturates and propofol, potentiate GABA responses in neurons. Higher concentrations, present in blood during total IV anesthesia, directly activate GABA-A receptors opening Cl- channels and thus reducing neuronal excitability. This represents a mechanism by which some IV GAs may achieve anesthesia. GABA-A receptors are heterogeneous consisting of five subunits. Each subunit could be one of fifteen varieties cloned so far. Using the patch- clamp technique to record GABA responses from single cells, GABA-A receptors with different subunit combinations have been found to possess distinct pharmacological properties. Preliminary data indicate that GABA-A receptor combinations, expressed by the neuronal GT1-7 cell line are not directly activated by high concentrations of IV GAs. To study the subunit specificity of GABA-A receptor modulation and activation by IV GAs, GABA-A receptor subunit cDNAs will be transfected into cell lines lacking endogenous receptors. The cells will first be transfected with homomeric receptors. These will be tested using the patch-clamp technique to determine whether homomeric receptors are functional (Specific Aim l). Having determined the subunits required for a receptor to respond to GABA, the subunits needed for potentiation of GABA-responses by IV GAs will be investigated (Specific Aim 2). If additional subunits are required for functional receptors, or modulation of GABA-A receptors by IV GAs, these will be co-transfected into cells. The subunits tested will be those for which mRNAs have been identified in GT1-7 cells using molecular techniques. Finally, recombinant GABA-A receptors will be tested to define the subunits required for direct activation by IV GAs (Specific Aim 3). In future, having established the subunit specificities for GABA-A receptor direct activation by GABA and IV GAs, and modulation by IV GAs, these will be compared to the subunit specificity for the similar actions of volatile agents at this receptor. An understanding of the subunit specificity of GABA-A receptor modulation by GAs and the distribution of these subunits in the brain may suggest areas involved in anesthesia. Once the target site(s) for GAs are defined it may be possible to develop more efficacious and safer agents.