Alpha subunit-dependent glycine receptor clustering and regulation of synaptic receptor numbers

A Patrizio,C G Specht,R Pizzarelli,M Renner,A Triller

doi:10.1038/s41598-017-11264-3

Abstract

Accumulation of glycine receptors at synapses requires the interaction between the beta subunit of the receptor and the scaffold protein gephyrin. Here, we questioned whether different alpha subunits could modulate the receptors’ diffusion and propensity to cluster at spinal cord synapses. Using quantitative photoactivated localisation microscopy we found that alpha-1 and alpha-3 containing glycine receptors display the same α3:β2 stoichiometry and gephyrin binding. Despite these similarities, alpha-3 containing receptors are less mobile and cluster at higher density compared to alpha-1, with 1500 versus 1100 complexes µm−2, respectively. Furthermore, we identified a subunit-specific regulation of glycine receptor copy numbers at synapses: when challenged with interleukin 1β, the synaptic occupancy of alpha-1 but not alpha-3 receptors was reduced. This mechanism may play a role in the cell-type dependent regulation of glycinergic currents in response to interleukin 1β and highlights the capacity of the alpha subunits to affect receptor-gephyrin binding at synapses.

Highlights

The clustering of inhibitory neurotransmitter receptors at synapses is largely dependent upon their interaction with the scaffold protein gephyrin
The pro-inflammatory cytokine interleukin 1β (IL-1β) causes rapid changes in glycinergic currents in different populations of spinal cord interneurons[14, 15]. In contrast to this view we hypothesise that the functional and dynamic properties of glycine receptor (GlyR) are not strictly separated between the α and the β subunits of the receptor. Such is the case for the GABAA receptor (GABAAR), where receptor diffusion is regulated in response to agonist binding and allosteric modulation[16]
We found clear evidence that α1 and α3 subunits modulate the dynamic equilibrium of GlyRs in spinal cord neurons even though they share the same heteropentameric stoichiometry and the same gephyrin binding capacity

Summary

Discussion

We tested the hypothesis that the GlyRα subunits influence the receptor clustering at synapses, which was thought to depend exclusively on the interaction of the β subunit with the gephyrin scaffold. We found that about 80 GlyR complexes are clustered at spinal cord synapses This compares to an average of 300 gephyrin molecules, meaning that GlyRs occupy up to half of the synaptic binding sites at steady state, considering their α3:β2 stoichiometry. We did observe that upon lentiviral expression of GlyRα subunits the immunoreactivity of endogenous gephyrin and GABAARβ3 was reduced compared to non-infected neurons (Figs S2 and S3) While this suggests that the GlyR occupancy of synaptic binding sites is altered in infected cells, it has no bearing on our quantification of absolute GlyR numbers, nor on the comparison between different α subunit variants and their regulation. Our results highlight the importance of studying defined neuron populations expressing known receptor subtypes

Methods

Findings

Additional Information