Molecular basis of the GABAA receptor {alpha}3 subunit interaction with gephyrin.

Tretter, V., Kerschner, B., Milenkovic, I., Ramsden, S.L., Ramerstorfer, J., Saiepour, L., Maric, H-M., Moss, S.J., Schindelin, H., Harvey, R.J., Sieghart, W. and Harvey, K. (2011) Molecular basis of the GABAA receptor {alpha}3 subunit interaction with gephyrin. Journal of Biological Chemistry . 10.1074/jbc.M111.291336 .

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DOI: 10.1074/jbc.M111.291336


The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N-terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3 and collybistin on gephyrin overlap. Curiously, two key residues (D327 and F330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit - E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μM and 0.2 μM, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3 and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.

Item Type:Article
Uncontrolled Keywords:GABAA receptor, GABRA3, gephyrin, synaptic clustering
Departments, units and centres:Department of Pharmacology > Department of Pharmacology
ID Code:2295
Journal or Publication Title:Journal of Biological Chemistry
Deposited By:Library Staff
Deposited On:06 Oct 2011 11:17
Last Modified:06 Oct 2011 11:17

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