Crosslinking/MS Studies of Cholesterol Interactions with Human α1 Glycine Receptor

Abstract

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) known to mediate fast neurotransmission throughout the brain and spinal cord. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. However, cholesterol at compositions below approximately 33 mol percent is reported to have negligible chemical activity. Taken together, the findings suggest that specific interactions between membrane proteins and cholesterol may become significant only at concentrations above this stoichiometric threshold, while interactions at lower cholesterol levels are primarily indicative of non-specific accessibility to the surrounding bilayer. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol:lipid ratios above and below the cholesterol activity threshold. Equivalent aliquots of azi-cholesterol, a photoactivatable cholesterol analog, were incorporated into reconstituted vesicles of varying cholesterol/lipid ratios. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsin digested and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by ESI-Q-TOF MS, and sites of cholesterol crosslinks in peptides were identified by targeted MS/MS. Differential sites of crosslinking as a function of cholesterol concentration were identified, with crosslinks found primarily in TM4 and extramembranous loops of GlyR. Concentration-dependent sites of interaction between cholesterol and GlyR were identified, highlighting sites of non-specific interactions of the receptor with its surrounding lipids, as well as potential specific cholesterol binding sites in GlyR.