Differential regulation of the postsynaptic clustering of γ-aminobutyric acid type A GABAA receptors by collybistin isoforms

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA A) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2 SH3+, CB2 SH3-, CB3 SH3+, and CB3 SH3-) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2 SH3- and gephyrin developed very large postsynaptic gephyrin and GABA A receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2 SH3+ and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2 SH3- or CB2 SH3+. Co-transfection with CB2 SH3- or CB2 SH3+ and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2 SH3- and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA A receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA A receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3- isoforms play a major role in the postsynaptic accumulation of GABA A receptors and in GABAergic synaptic strength. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc