Examining glycine receptor's intracellular domain by crosslinking-mass spectrometry

Duquesne University received a $512,397 Project Grant award from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859) to conduct crosslinking-mass spectrometry (CXMS) studies examining the intracellular domain (ICD) structure of glycine receptors. The three-year project, initiated September 11, 2025, and concluding August 31, 2028, will employ complementary analytical techniques to characterize full-length A1 glycine receptors in reconstituted lipid bilayers across different allosteric states, with emphasis on the previously unresolved ICD.

The research will deliver two primary scientific outcomes: Specific Aim 1 will generate structural data on the ICD through CXMS analysis using variable-length photoactivatable protein-protein crosslinkers distributed across the ICD as a function of glycine receptor allostery; Specific Aim 2 will characterize lipid-protein interactions using lipid crosslinkers with photoactivatable moieties at varying bilayer depths, focusing on ICD associations with the lipid bilayer. Collectively, these approaches will provide structural insights into the glycine receptor's intracellular domain that complement high-resolution structural studies conducted in membrane-mimetic environments, advancing fundamental understanding of pentameric ligand-gated ion channel biology relevant to neurological disease research and treatment development.

Abstract

While high-resolution structural data are available for members of the pentameric ligand-gated ION channel (plgic) superfamily in membrane-mimetic environments, the structure of the intracellular domain (ICD) of these receptors in lipid bilayers remains unresolved. Our aims are to conduct crosslinking mass spectrometric studies (cxms) to complement current methods and characterize the structure of full-length A1 glycine receptors (glyrs) in reconstituted bilayers different allosteric states, with a particular focus on the ICD. Specific aim 1 will use cxms to interrogate the protein structure of the ICD around single sites of attachment of a variable-length photoactivatable protein-protein crosslinkers distributed over the full length of the ICD as a function of glyr allostery. Specific aim 2 will use cxms to examine lipid-protein interactions as a function of allostery using lipid crosslinkers with their respective photoactivatable moieties at different depths of the bilayer, with a particular focus on the close associations of the ICD with the lipid bilayer. Together, the cxms approaches used in each aim will provide insight into the structure of this previously unresolved domain and complement high resolution studies conducted in membrane-mimetic environments.