Receptors, gephyrin and gephyrin-associated proteins: novel insights into the assembly of inhibitory postsynaptic membrane specializations

Abstract

The synaptic localization of ion channel receptors is essential for efficient synaptic trans-mission and the precise regulation of diverse neuronal functions, such as signal integration and synaptic plasticity. Emerging evidence points to an important role of cytoskeleton-associated proteins that assemble receptors and components of the subsynaptic machinery at postsynaptic membrane specializations. This article reviews interactions of inhibitory postsynaptic neurotransmitter receptors with the receptor anchoring protein gephyrin and intracellular components involved in downstream signalling and/or control of signal transduction processes. The presently available data suggest a central synaptic organizer function for gephyrin in inhibitory postsynaptic membrane assembly and stabilization.

Figure 1. A model for activity-dependent GlyR clustering at developing postsynaptic membrane specializations

A glycinergic presynaptic terminal and a segment of the plasma membrane of a GlyR-expressing postsynaptic neuron are shown at different stages of synaptogenesis. A, immature stage: GlyRs are randomly distributed in the plasma membrane of the postsynaptic cell, whereas gephyrin is cytoplasmically localized. B, initiation of gephyrin cluster formation: release of glycine from the presynaptic terminal gates neighbouring GlyRs. This results in a depolarizing Cl⁻ efflux (downward arrows) that activates nearby voltage-dependent Ca²⁺ channels. The resultant local microdomain of elevated Ca²⁺ concentration triggers the membrane apposition and aggregation of gephyrin at sites of GlyR activation. C, maturation stage: the growing submembraneous gephyrin aggregate traps additional GlyRs underneath the presynaptic terminal and immobilizes the receptors in the developing postsynaptic membrane by anchoring them to the subsynaptic cytoskeleton. Due to a change in Cl⁻ equilibrium potential, GlyR activation triggers Cl⁻ influx causing hyperpolarization (upward arrows), and thus inhibition of neuronal firing. This model is based on data by Kirsch & Betz (1998) and does not include G protein-mediated signalling pathways that may also contribute to the clustering process (Kins et al. 2000).

Figure 2. GABA_ARs require gephyrin for synaptic cluster formation

Immunochemistry of cultured hippocampal neurons from wildtype +/+ (A) and geph -/- (gephyrin-deficient) mice (B). After 21 days in vitro, the cultures were stained with antibodies specific for the GABA_AR subunit α2. Note the loss of GABA_A receptor clusters in neurons from geph -/- mice. These mutant neurons consistently show a significant increase in intracellular GABA_A receptor subunit immunoreactivity. Scale bar, 20 μm; applies to both A and B. (Modified from Kneussel et al. 1999a.)

Figure 3. Schematic representation of postsynaptic gephyrin and its binding partners

Gephyrin is depicted as a submembraneous protein scaffold that concentrates structural and membrane components at the postsynaptic membrane. Gephyrin directly binds and clusters GlyRs and is essential for the postsynaptic localization of a major population of GABA_ARs; these GABA_ARs may use the tubulin binding protein GABARAP for interaction with gephyrin. Gephyrin also binds to phosphatidylinositol 3,4,5-trisphosphate (PIP₃) binding proteins involved in actin dynamics and downstream signalling, such as collybistin and profilin, and interacts with RAFT1 (rapamycin and FKBP12 target protein), a candidate regulator of dendritic protein synthesis. Additionally, gephyrin catalyses a crucial step in the biosynthesis of the molybdenum cofactor (MoCo); whether this catalysis also occurs at the synapse is presently unclear. The tubulin binding properties of gephyrin closely resemble those of MAP1-B, a protein thought to link GABA_C receptors to microtubules and ensure tight anchoring of both GlyRs and GABA_A receptors to subsynaptic microtubules.