Identification and subunit composition of GABA-A receptor subtypes

A receptor subtypes Research project P 14385 Identification of GABA A receptor subtypes Dr. Werner Sieghart 08.05.2000 Most of the actions of g -aminobutyric acid (GABA), the major inhibitory transmitter of the central nervous system, are mediated by GABA A receptors. GABA A receptors are chloride ion channels that can be opened by GABA and are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anesthetics and convulsants. GABA A receptors consists of fife subunits usually belonging to three different subunit classes. So far, 6a , 3b . 3g , 1d , 1e , 1p , 1q and 3r subunits have been identified in the brain that could give rise to a large number of GABA A receptor subtypes with distinct subunit composition. How many GABA A receptor subtypes actually are formed in the brain presently is not known and only a few of these subtypes have been identified. This project aims to identify a large part of all GABA A receptor subtypes present in the brain and to determine their relative abundance by using a subtractivee purification method recently developed by our group. For this, GABA A receptor subtypes containing a certain subunit will be completely eliminated from brain membrane extracts by immunoaffinity chromatography on subunit specific antibodies. The subunit composition of receptors eliminated will then be determined by Western bolt analysis. Measurement of GABA A receptors present in the extract and column efflux will provide information on the percentage of receptors removed and thus, on the relative abundance of the respective receptor subtypes. Subunits copurifying with the eliminated subunit will then be eliminated one-by-one unsing the corresponding immunoaffinity columns and the percentage of receptors removed containing other subunits will be determined. These studies willprovide information on the extent of colocalization of various GABA A receptor subunit pairs in the same receptor. The complete sunbunit composition and relative abundance of the corresponding receptor subtypes will then be determined after elimination of all the other receptors by suitable immunoaffinity columns. Information on GABA A receptors subtypes expressed in certain brain tissues can be used for the development of receptor subtypes-selective drugs and will supplement immunocytochemical studies on the colocalization of individual subunits. In addition, detailed knowledge on receptor composition is essential for future studies investigating changes in GABA A receptor subunit composition in the course of chronic drug treatment or of certain diseases such as epilepsy, stroke or neurodegenerative disorders.

Gamma-aminobutyric acid (GABA) is the major inhibitory transmitter of the central nervous system. Most of its actions are mediated by GABA A receptors. GABA A receptors are chloride ion channels that can be opened by GABA and are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anesthetics and convulsants. GABA A receptors consist of five subunits usually belonging to three different subunit classes. A variety of subunits from different subunit classes have been identified in the brain that could give rise to a large number of GABA A receptor subtypes with distinct subunit composition. How many GABA A receptor subtypes actually are formed in the brain presently is not known and only a few of these receptor subtypes have been identified. In the course of this project, for the first time the subunit composition and quantitative importance of major and minor GABA A receptor subtypes were determined in mouse and rat cerebellum. In addition, GABA A receptor subunit expression was investigated in various brain regions of mice, in which a GABA A receptor subunit was either deleted or overexpressed. Results indicated that deletion as well as overexpression of subunits causes changes in the expression of other subunits and in receptor subunit-composition. From the changes observed, conclusions on possible subunit partnerships within receptors and on mechanisms of receptor assembly could be derived. The applied techniques can now be used to study changes in the expression of GABA A receptor subtypes in the course of various diseases of the nervous system. Information on GABA A receptor subtypes expressed in certain brain tissues as well as on changes in their subunit composition during diseases can be used to define targets for the development of receptor subtype-selective drugs and thus, will stimulate innovation and competitivity in the pharmaceutical industry. Finally, in an international collaborative study a mouse model was developed and investigated, that will allow us to rapidly and selectively enhance or reduce the electrical activity of certain neuronal cell types in the brain by the application of ligands interacting with the benzodiazepine binding site of GABA A receptors. These mice will be used to study the consequences of such manipulations on network-oscillations in the brain as well as on behaviour of the mice. This strategy will lead to a major breakthrough in brain research because it will teach us how individual nerve cell types and their receptors contribute to the overall functioning of the brain and to the generation of behaviour, learning and memory.