Functional and structural characterisation of GABAA receptor clustering proteins

摘要

GABAA receptors (GABAAR) are the major inhibitory neurotransmitter receptors in the human brain. The scaffolding protein gephyrin clusters GABAAR at postsynaptic sites through a direct interaction with the GABAAR alpha1-3 subunits. The guanine nucleotide exchange factor collybistin facilitates translocation of gephyrin to postsynaptic sites. Symptoms of impaired GABAAR function include epilepsy and intellectual disability. The aim of my project was to further understanding of the clustering mechanisms of GABAAR using structural and molecular biology. I previously identified collybistin genes and describe here the identification of GABAAR genes in zebrafish using bioinformatics. To better understand the role of gephyrin in GABAAR clustering I purified gephyrin protein from different species for crystallisation. As partial structures for the N- and C-terminal domains are published my aim was to crystallise full-length gephyrin and the C-terminal E-domain with and without GABAAR alpha2 or alpha3 subunits. I obtained gephyrin E-domain crystal structures with a resolution of 2.3A and 3.0A. The improvement on the resolution and a novel space group to available E-domain structures allowed the observation of differences especially in the flexible subdomain II. Lastly, two novel collybistin mutants reported in patients with intellectual disability were analysed. Collybistin mutants R290H and R356Q showed no altered gephyrin protein interaction strength in comparison to wild- type but lost the ability to bind phosphatidylinositol 3-phosphate (PI3P) in binding assays. In addition, co-expression of collybistin mutants with gephyrin in mammalian cells showed the loss of gephyrin submembrane microcluster formation. Modelling using the published collybistin crystal structure suggested that the loss of a hydrogen bond in mutant R290H destabilise the protein structure, whereas for mutant R356Q, the substituted arginine seems to be essential for PI3P binding. In conclusion, my work provides some new insights into the role of GABAA receptor clustering proteins in health and disease.