Yuan, Banghao; (2023) Regulation of GABA(A) receptor ER-to-Golgi trafficking by the ER-resident chaperones. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Type A γ-aminobutyric acid receptors (GABAARs) represent a family of pentameric GABA-gated Cl-/HCO3- ion channels which mediate the inhibitory neurotransmission in the brain. The appropriate assembly of the receptor subunits and their transient interactions with chaperones within the endoplasmic reticulum (ER) and Golgi apparatus are essential for the GABAARs intracellular trafficking which regulates cell-surface expression of the receptors to fulfil their function. I started my research with characterising the components and structures of Hsp90 cytoplasmic complexes through chemical cross-linking and mass spectrometry. Following this, I have focused on ER-resident Hsp90 homologue, Grp94, in GABAAR intracellular processing. Mutations in the α1, β3 and γ2 subunits at a highly conserved region located at the end of the N-terminal extracellular domain adjoining the transmembrane region 1 were shown to impair GABAAR cell-surface expression in heterologous cell systems. Using co-immunoprecipitation and immunocytochemistry, I have demonstrated that mutated receptor subunits are retained in the ER, where they associate with Grp94 that has been reported to regulate the ER-associated degradation of the α1 subunit. When compared to the wt receptors, mutated receptors show increased association with Grp94. However, Grp94 inhibitor, PU-WS13, could not rescue mutated GABAARs cell-surface expression, and when Grp94 is overexpressed, the amount of wt GABAAR did not change. Structural modelling using Modeller and Molsoft-ICM revealed the significance of this conserved region in maintaining the structural stability of the N-terminal domain outer β-sheet through the network of hydrogen bonds. This region is shown to be important for calnexin binding to the glycosylated asparagine residue N174. The structural alterations caused by mutations leading to reduced calnexin binding to GABAARs in biochemical experiments indicate that this previously uncharacterised region plays an important role in stabilising GABAAR interactions with calnexin in the ER and thus regulate the processing and trafficking of these receptors to the cell-surface.

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