A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin

Introduction: The inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring.Objectives: We identified a poly-proline II helix (PPII) in position 365-373 of the intra-cellular TM3-4 loop of the human GlyR alpha 1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested.Methods: Glycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments.Results: Recombinantly expressed collybistin bound to isolated GlyR alpha 1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyR alpha 1(P1)(-)(5A)) located in the GlyR alpha 1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyR alpha 1(P1)(-)(5A) mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyR alpha 1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyR alpha 1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyR alpha 1(P366L), identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding.Conclusion: Our data suggest a novel interaction between alpha 1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.