Differential Regulation of the Postsynaptic Clustering of γ-Aminobutyric Acid Type A (GABAA) Receptors by Collybistin Isoforms

Abstract

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and ␥-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 nontransfected 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.A fundamental issue in the GABAergic synapse field is to understand the mechanisms that regulate the postsynaptic clustering of GABA A 3 receptors (GABA A Rs) and GABAergic synaptic strength during inhibitory synapse formation. Collybistin (CB) is a cytoplasmic protein that binds to gephyrin, helping the latter to cluster and translocate to the submembranous compartment (1-4). CB is a guanine nucleotide exchange factor (GEF) that catalyzes GDP-GTP exchange on the small GTPase Cdc42 of the Rho family (5). CB is essential for the initial synaptic localization and maintenance of gephyrin and GABA A Rs at GABAergic synapses in the hippocampus and amygdala (6, 7).In adult rat or mouse brain, two alternative spliced forms are expressed, CB2 and CB3, which are identical except for the C termini (3). There is also CB1 with a different C terminus, but this isoform is not expressed in neurons or in the adult brain. In humans, CB3 is called hPEM2. However, CB2 has not been detected in humans (3). There are also splice variants of CB2 and CB3 (or hPEM2) with or without an Src homology 3 (SH3) domain (1, 3). Alt...

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses

“…Similarly, overexpression of the SH3 domain-deficient Cb isoform, which strongly binds PI3P (9 -11), causes increases in the density and size of perisomatic gephyrin clusters (49,50). This indicates that the interaction of Cb and gephyrin with PI3P-containing endosomes is of particular importance for the regulation of the size of perisomatic synapses, which typically produce a stronger inhibitory drive than the smaller synapses localized on dendrites (51).…”

γ-Aminobutyric Acid Type A (GABAA) Receptor α Subunits Play a Direct Role in Synaptic Versus Extrasynaptic Targeting

“…NL2 overexpression may also change GABA A -R subunit composition as shown in cerebellar granule cells (74). Collybistin can facilitate gephyrin localization to submembrane sites (75) and increase synaptic GABA A -R accumulation (76). Collybistin deficiency results in region-specific loss of gephyrin and a subset of GABA A -Rs, as well as altered synaptic plasticity and increased levels of anxiety (77,78).…”

Molecular and functional heterogeneity of GABAergic synapses

“…Insight into the roles of CB isoforms for gephyrin clustering was derived from over-expression studies in transfected neurons, which aimed to distinguish between CB2 SH3+ and CB2 SH3− [50,46], as well as to elucidate the contribution of Cdc42, the only known substrate of CB. In both studies, CB over-expression contributed to stabilize gephyrin, as evidenced by a marked increase in size and density of postsynaptic gephyrin clusters.…”