Molecular determinants of GABAA receptor degradation.

y-amino-butyric acid type A (GABAA) receptors are the major sites of fast synaptic transmission in the central nervous system and can be assembled from 7 subunit classes: al-6, (31-3, yl-3, 5, e, n, and 6. Although expression of receptor a and (3 subunits can produce functional GABA-gated chloride channels the presence of the y2 subunit within the GABAA receptor complex has been shown to play a key role in clustering and synaptic targeting. The regulation of GABAA receptor cell surface stability by endocytosis has been previously shown to be a crucial determinant of inhibitory synaptic strength. The main focus of this thesis is to further understand the molecular mechanisms by which the number of GABAA receptors clustered in synapses can be regulated. Microscopy and biochemical experiments showed that the y2 subunit of GABAA receptors confers an enhancement in the targeting of GABAA receptors to a degradative pathway after internalization from the plasma membrane. Furthermore, blocking lysosomal degradation with the lysosomal inhibitor leupeptin results in increased GABAergic currents in cells expressing GABAA receptors containing ocl, (33 and y2 subunits but not those expressing a 1(33 receptors alone. In order to characterise the molecular signals determining this degradative pathway, (33-y2 chimeras were created and expressed in both HEK293 cells and hippocampal neurons. Quantitative confocal microscoscopy studies on these chimeras revealed a 20 amino acid region to be responsible for the late endosomal/lysosomal targeting of y2 subunit containing GABAA receptors. Further analysis into this 20 amino acid region identified a lysine stretch that when mutated resulted in decreased levels of internalised GABAA receptors in late endosomes. Within this region the mutation of serine 327, a known phosphorylation site of GABAA receptors, also caused a reduction in the targeting of internalised GABAA receptors to lysosomes. Interestingly GABAergic currents formed by the expression of a 1(33 and y2L (lysine or serine mutant) were unaffected by the lysosomal inhibitor leupeptin. Biochemical studies found these lysines to be modified by the small poly-peptide ubiquitin, a known molecular signal for the degradation of proteins in the proteasome and the endocytic pathway. Moreover, mutation of serine 327 was sufficient to inhibit the ubiquitination of this lysine stretch in the intracellular loop of the y2L subunit. The results presented in this thesis suggest that the endocytic sorting fate of synaptic GABAA receptors plays an important role in determining the strength of inhibitory synapses and provide a mechanism by which serine 327 acts as a molecular switch to regulate the ubiquitination dependent degradation of GABAA receptors in the lysosome