Identification of amino acid residues contributing to the formation of the benzodiazepine binding pocket
Several amino acid residues were identified that are involved in the formation of the benzodiazepine binding pocket of GABAA receptors. In addition, the relative orientation of ligands and amino acid side chains in the benzodiazepine binding pocket of GABAA receptors was for the first time determined. This information is of interest for basic scientists investigating the structure and function of GABAA receptors and is also of interest for industry. It can be used to design receptor models that might help in a more rational development of drugs fighting human diseases.
Light and electron microscopic techniques as well as electrophysiological methods were used to establish the function of individual synapses on identified neurons. A calibration was achieved that relates the number of GABAA receptors present on the synapses to the electrophysiological signal transmitted and this opened a novel way of quantitative comparison of individual synapses. The finding that different synapses on the same neuron might contain different amounts of the same receptor or might contain different GABAA receptor subtypes, indicates that neurons in the brain are able to selectively target GABAA receptors to individual synapses depending on the source of input on the same cellular domain. In addition, the interconnections between GABAergic neurons in the hippocampus and cerebral cortex were investigated and quantitatively defined. These structures are important for cognition, memory and learning. Results obtained have led to the formulation of a conceptual framework of the synaptic organization of the cerebral cortex which will facilitate progress in the understanding of this complex neuronal network. These results are of interest for basic research concentrating on the structure and function of the brain and provide the basis for the understanding of complex electrical phenomena in this tissue.
An efficient and practical route for the synthesis of simplified bicuculline analogues was established and allowed to synthesize and identify two new classes of compounds that enhance GABA-induced chloride ion flux. One of these classes interacts via the benzodiazepine site and one of these classes enhances GABA-induced chloride ion flux by interacting with a novel modulatory site on GABAA receptors. . Each class of compounds exhibits some receptor subtype selectivity. In each of these classes positive and negative allosteric modulators were identified. In addition, some of these compounds exhibit antagonist properties and inhibit the actions of other compounds of the same class. Since all of these compounds don't directly activate GABAA receptor associated chloride ion channels, they presumably exhibit low toxicity. Compounds from each of these classes might have therapeutic potential for treatment of anxiety, convulsions, sleep disorders, and cognitive dysfunctions. Since some of these compounds do not interact with the benzodiazepine binding site of GABAA receptors, they might also not induce the development of tolerance observed with benzodiazepines on long term anticonvulsive treatment. The partners currently are looking for industrial partners interested in a clinical development of these compounds.
A generally applicable method for the determination of the sub-unit composition and quantitative importance of heterooligomeric receptors was developed. The method is based on a subtractive purification procedure using immunoaffinity chromatography of detergent-extracted receptors. Applying this method, all GABAA receptor subtypes present in the brain (or other hetero-oligomeric receptors, if antibodies are available) can be identified and their sub-unit composition can be determined. In addition, changes in the sub-unit composition as a consequence of diseases, chronic drug treatment and sub-unit knockouts can be measured. Here, the method was used to determine the composition of various GABAA receptor subtypes in the brain. In addition, the regional distribution of 13 different GABAA receptor sub-units was established in the brain and the cellular and sub-cellular localization of several of these sub-units was determined using light and electron microscopic techniques. These results are important for basic research and industry. They provide information on the sub-unit composition of GABAA receptors in tissues that are targets for drug development. Knowledge on receptors actually occurring in the target tissue allows to select the corresponding recombinant receptors as a tool for the development of receptor subtype selective drugs.