Kittler, Josef Thomas Jacques; (2000) Molecular mechanisms of GABAA receptor anchoring and trafficking. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

γ-Aminobutyric acid type A (GABAA) receptors are members of the ligand gated ion channel superfamily and are the major sites of fast synaptic inhibition in the brain. GABAA receptors are clustered at inhibitory synapses, however, precisely how these receptors are inserted into postsynaptic membranes and stabilised at synapses remains unknown. The focus of this thesis is to further understand the mechanisms important for the targeting and cell surface stability of ionotropic GABAA receptors. To examine the membrane trafficking of these receptors a fusion protein of the γ2L subunit with green fluorescent protein (GFP) was produced. This fusion protein was functional and allowed the visualisation of receptor membrane targeting and endocytosis in live cells. Interestingly, the targeting of this construct to GABAergic synapses in cultured hippocampal neurones was dependent upon coassembly with receptor α and β subunits. This suggests that the assembly and membrane targeting of GABAA receptors containing the γ2 subunit follow similar itineraries in heterologous systems and neurones. GABAA receptor β and γ subunits were also found to associate with the adaptin complex AP2, a protein implicated in the recruitment of transmembrane proteins into clathrin coated pits. Furthermore, in cultured hippocampal neurones blocking dynamin-dependant endocytosis caused a gradual increase in the amplitude of mIPSCs that reached a plateau at 1.7 to 2.3-fold of it's control value within 40-50 min. These results suggest there must be a relatively fast turnover of GABAA receptors between the cell surface and intracellular compartments. To further study the role of the GABAA receptor binding partner, GABARAP, we compared the subcellular distribution of this protein with that of gephyrin, a protein important for GABAA receptor synaptic clustering. In cultured hippocampal neurones GABARAP associated weakly with synaptic GABAA receptor and gephyrin clusters. However, GABARAP was found at high levels on intracellular membrane compartments including the Golgi and subsynaptic cisternae. Furthermore, GABARAP was found to associate with N-ethylmaleimide sensitive factor (NSF), a protein essential for intracellular membrane transport events and important for receptor trafficking. This suggests that GABARAP may act to facilitate the trafficking of GABAA receptors via it's ability to interact with NSF. The results presented in this thesis suggest that GABAA receptors cycle into and out of the plasma membrane in a process that may be regulated by their association with adaptin complexes and recruitment of NSF via GABARAP. This may allow neurones to specifically modify cell surface receptor levels which may have a critical role in controlling the efficacy of inhibitory synaptic transmission.

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