Oliver, Harriet Elizabeth Warwick; (2021) Subcellular localisation of α3-GABAA receptors: a role for phosphorylation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

GABAA receptors are the primary mediators of inhibitory neurotransmission in the brain. In contrast to most αβγ-GABAA receptor synaptic-subtypes, α3 subunit-containing receptors localise in both synaptic and extrasynaptic locations, where they mediate phasic and tonic inhibition respectively. Of particular note is the synaptic localisation of α3-GABAA receptors in the thalamic reticular nucleus, a brain region linked to absence epilepsy; and the extrasynaptic localisation of α3-GABAA receptors in the basolateral amygdala, a region involved in the expression of anxiety and fear disorders. To date, our understanding of the molecular mechanisms that control the subcellular localisation of the α3-GABAA receptor remains limited, including its interactions with the synaptic scaffold protein gephyrin, which tethers GABAA receptors at inhibitory synapses. In this study, we investigated the role that phosphorylation, a common post-translational modification that affects receptor trafficking, plays in the regulation of α3-GABAA receptor subcellular localisation. Phospho-null (phenyl)alanine and phospho-mimetic aspartate mutations were used to manipulate the phosphorylation states of three key residues – T400, T401 and Y402 – in the gephyrin binding domain of α3. Electrophysiological interrogation of these phospho-mutants in HEK-293 cells and cultured hippocampal neurons revealed alterations in receptor GABA sensitivity and kinetics, consistent with changes in subcellular localisation, when phosphorylation was mimicked at Y402. Further investigation using structured illumination microscopy demonstrated a reduction in the proportion of inhibitory synapses containing α3Y402D-GABAA receptors, but these receptors were more concentrated in those synapses where they were retained. This concentration appears to be mediated through gephyrin-independent mechanisms, as co-localisation between α3Y402D-GABAA receptors and gephyrin was reduced compared to phospho-null and wildtype receptors. These data indicate that phosphorylation of Y402 affects the subcellular localisation of α3-GABAA receptors. This study provides a basis for further investigation of the role phosphorylation plays in receptor localisation in distinct brain regions, and how this may be manipulated for therapeutic benefit.

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