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Dissecting the structural role of GABAA receptors in synapse formation

Brown, LE; (2016) Dissecting the structural role of GABAA receptors in synapse formation. Doctoral thesis , UCL (University College London).

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Abstract

GABAergic synapse formation involves the establishment of specific cellcell contacts between the presynaptic GABAergic neurones and their postsynaptic targets. Postsynaptic GABAA receptors (GABAARs) themselves have been shown to play a direct structural role in the initiation of functional synapses (Fuchs et al., 2013). Yet, characterisation of the molecular mechanisms underlying their structural involvement remains sparse. As the large N-terminal extracellular domains (ECDs) of the GABAAR subunits reside within the synaptic cleft, we hypothesised that these domains participate in trans-synaptic interactions with cleft-spanning presynaptic proteins. To investigate our hypothesis, a baculovirus/Sf9 cell expression system was used to express and purify the ECDs of the α1, β2 and γ2 GABAAR subunits. These ECDs were utilised in proteomics and mass spectrometry experiments to identify candidate trans-synaptic interacting proteins. To dissect the structural role(s) played by individual GABAAR subunits in synapse formation, a co-culture model system incorporating GABAergic medium spiny neurones and HEK293 cells expressing different combinations of GABAARs was developed. The results indicated that the γ2 subunit was necessary for contact formation and that its ‘synaptogenic’ effects were influenced by the subtype of α and β GABAAR subunits that were incorporated within the receptor. To elucidate whether the synaptogenic effects of the GABAAR subunits were directly mediated by their ECDs, the purified α1, β2 and γ2 ECDs were added to HEK293-MSN co-cultures at 14 Day in vitro (DIV). Contact formation was reduced in the presence of each of the exogenous ECDs. In parallel, the ECDs were added to pure cultures of MSNs to analyse their structural effects on GABAAR cluster size and number. The results demonstrated that synapsespecific effects were observed for each ECD. In summary, the synaptogenic effects of GABAARs depend on their individual subunit composition, with the ECDs structurally contributing towards the initial cell-cell contacts.

Type: Thesis (Doctoral)
Title: Dissecting the structural role of GABAA receptors in synapse formation
Event: UCL (University College London)
Language: English
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
URI: http://discovery.ucl.ac.uk/id/eprint/1476757
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