Saiepour, Leila; (2009) Subunit-selective interactions of GABAA receptors with the clustering proteins gephyrin and collybistin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory receptors in the central nervous system. These pentameric receptors contain different subunits, with the most common subtypes consisting of two α, two β and a single γ2 subunit. Changes in GABAAR expression, cellular distribution and function lead to neurological disorders including epilepsy, anxiety and depression. Postsynaptic clustering of GABAARs is thought to be important for their correct function, but the exact mechanisms involved remain unclear. A key molecule involved in the synaptic localisation of GABAARs is the tubulin linker protein gephyrin, as demonstrated by the loss of most synaptic GABAARs in gephyrin knockout mice. Recently, a direct interaction between gephyrin and the GABAARs α2 subunit was shown. However, other studies have implicated the involvement of an alternatively spliced isoform of the γ2 subunit (γ2L) in GABAAR clustering. In addition, knockout mice for the GDP/GTP exchange factor collybistin, which facilitates the correct targeting of gephyrin to postsynaptic membranes, also resulted in the loss of GABAAR clustering in several brain regions. I have examined the potential role of GABAAR α-, β- and γ-subunits in postsynaptic receptor clustering using a range of multidisciplinary techniques. Using the yeast-two hybrid system, I have demonstrated a direct interaction between selected GABAAR α subunit intracellular M3-M4 loops with subdomains of gephyrin and collybistin. Interestingly, I observed an enhanced interaction between gephyrin and the GABAAR α2 subunit in the presence of collybistin in a yeast tri-hybrid system, suggesting the formation of a complex containing these three proteins. However, I have also found that the M3-M4 loop of the GABAAR γ2L subunit localises to the submembrane compartment in mammalian cells, where it co-localises with gephyrin/collybistin aggregates. In conclusion, my data suggest a complex regulation of GABAAR clustering involving multiple GABAAR subunits and additional membrane-associated proteins.

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