Cellular distribution and immobilisation of GABA(_A) receptors

Abstract

Synaptic inhibition in the vertebrate central nervous system is largely mediated by type A GABA receptors (GABA(_A)R). The clustering of (GABA(_A)R) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To discern the role that specific GABA(_A)R subunits play in determining the receptor's cell surface topography and mobility, recombinant GABA(_A)Rs, comprising different GABA(_A)R subunit combinations, were transiently expressed in COS7, HEK293 and PC12 cells. In addition, a series of domain swapping experiments were performed in order to elucidate which regions of the protein are important in mobility/anchoring of receptors. The cellular localization and lateral mobility of the recombinantly expressed GABA(_A)Rs were determined by immunocytochemistry and Fluorescence Photobleach Recovery (FPR), respectively. The results presented in this thesis show that GABA(_A)R al subunits are recruited by the β3 subunits from an internally sequestered pool and assembled into a population of GABA(_A)Rs that are spatially segregated into clusters and also immobilised on the cell surface. FPR experiments on recombinant GABA(_A)R containing al-a6 subunits expressed in COS? cells showed restricted mobilities consistent with mobility constants determined for native GABA(_A)Rs expressed on cerebellar granule cells. Furthermore, the intracellular loop domain M3/M4 of the a1 subunits was found to be required for anchoring recombinantly expressed GABA(_A)Rs in C0S7 and cerebellar granule cells in culture, but not for GABA(_A)R clustering at the cell surface.