Investigation of the intersubunit contacts governing assembly of GABAA receptors

GABA A receptors are the quantitatively most important inhibitory receptors in the central nervous system, and are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics and convulsants. GABA A receptors are choride ion channels that can be opended by gamma-aminobutyric acid (GABA), and are composed of 5 protein subunits. In this project for the first time amino-acid sequences of GABA A receptor subunits were identified that are responsible for the formation of direct contacts with neighbouring subunits. In addition, it was demonstrated that some of these sequences are responsible for the formation of very specific contacts with certain subunits only, and thus influence the subunit arrangement within the receptors. Finally, it was shown that in different subunits homologous amino acid sequences are responsible for the formation of intersubunit contacts and that these sequences are located at comparable positions in different subunits. Results from this project not only explain how the subunit arrangement within the receptors is generated, but also identify the location of certain amino acid residues that are important for the formation of pharmacological binding sites of these receptors. Aims of the project: This project aimed to identify amino acid sequences of GABA A receptor subunits that are responsible for the formation of specific contacts between different subunits of this receptor and by that, influence receptor assembly as well as the arrangement of the subunits. This information is important for all further investigations on the structure and function of these pharmacologically and clinically important receptors, and can be directly incorporated into structural models of GABA A receptors and their pharmacological binding sites. Results of this project: Work on this project has identified a total of two different amino acid sequences on the beta3 subunit, and a total of three amino acid sequences each on the gamma2, gamma3, and alpha1 subunit, that are important for assembly with neighbouring subunits. In addition, for the first time a direct contact could be demonstrated between one of the identified alpha1 and gamma2 sequences. Since in some of these sequences amino acid residues are present that contribute to the formation of the GABA or benzodiazepine binding pocket, these sequences could be located at the respective interfaces that are involved in forming these pockets. These results also indicated that in different subunits homologous sequences are important for the formation of intersubunit contacts, and that some of these sequences cause interaction with some, but not all of the other subunits, and thus influence the arrangement of subunits within the receptors. Possible applications and consequences: Work on this project for the first time led to structural information on amino acid sequences important for direct contacts between GABA A receptor subunits. Since important pharmacological binding sites are located at the interfaces of these receptors, this information can be directly used for modeling of receptors and binding sites. An exact knowledge of the structure of these binding sites can be used for a more rational development of drugs with less side effects. Such drugs will have important clinical applications.