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A gephyrin-related mechanism restraining glycine receptor anchoring at GABAergic synapses.

ABSTRACT: Spinal cord neurons release glycine and GABA and accumulate glycine receptors (GlyRs) and GABA(A) receptors in the same postsynaptic densities. In contrast, supramedullar neurons prefer GABA as a neurotransmitter and exclude GlyRs from postsynaptic anchoring. The general aim of the present study was to elucidate the mechanisms underlying transmitter-appropriate receptor accumulation at inhibitory synapses. Specifically, we intended to clarify the molecular basis for the prohibition of GlyR accumulation in the postsynaptic densities of GABAergic synapses. A green fluorescent protein (GFP)-tagged gephyrin-binding loop of the GlyR beta subunit (GFP::betaL) was used as a surrogate for full-length receptors to characterize the GlyR binding capacity of postsynaptic gephyrins in transfected neurons. Both in spinal cord neurons (SCNs) and hippocampal neurons (HNs) GFP::betaL distribution displayed transmitter specificity; i.e., postsynaptic accumulation of GFP::betaL was high opposite terminals able to release glycine and low opposite purely GABAergic terminals. When comparing SCN and HN cultures we found that the level of mRNA coding for gephyrin splice variants containing the cassette C5 (C5-gephyrins) was significantly higher in HNs. In HNs depleted of C5-gephyrins, both GFP::betaL and endogenous GlyRs accumulated at postsynaptic GABAergic sites. Accordingly in SCNs, GFP-tagged C5-gephyrin displayed a preferential postsynaptic accumulation opposite GABAergic synapses. Comparison of glycinergic, mixed, and GABAergic synapses in SCNs showed that the degree of GlyR accumulation was inversely related to the amount of postsynaptic C5-gephyrin. These results identify the C5 splice variant of gephyrin as a factor regulating the transmitter-appropriate degree of GlyR accumulation at inhibitory synapses.

SUBMITTER: Meier J

PROVIDER: S-EPMC6730342 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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A gephyrin-related mechanism restraining glycine receptor anchoring at GABAergic synapses.

Meier Jochen J Grantyn Rosemarie R

The Journal of neuroscience : the official journal of the Society for Neuroscience 20040201 6

Spinal cord neurons release glycine and GABA and accumulate glycine receptors (GlyRs) and GABA(A) receptors in the same postsynaptic densities. In contrast, supramedullar neurons prefer GABA as a neurotransmitter and exclude GlyRs from postsynaptic anchoring. The general aim of the present study was to elucidate the mechanisms underlying transmitter-appropriate receptor accumulation at inhibitory synapses. Specifically, we intended to clarify the molecular basis for the prohibition of GlyR accum ...[more]

PMID: 14960612

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Project description:Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.

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A gephyrin-related mechanism restraining glycine receptor anchoring at GABAergic synapses.

Publications

A gephyrin-related mechanism restraining glycine receptor anchoring at GABAergic synapses.

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