Concentration-Dependent Differential State Crosslinking of azi-Cholesterol with Human Alpha 1 Glycine Receptor using Mass Spectrometry

Abstract

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. 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. Cholesterol at compositions below ∼33 mol% has negligible chemical activity. Taken together, the findings suggest that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold, while interactions at lower cholesterol levels will be 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 with equivalent aliquots of azi-cholesterol. State-dependent crosslinking of the resting and desensitized states was achieved by the absence and inclusion of saturating concentrations of glycine (10 mM), respectively. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsinized and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by ESI-Q-TOF MS, and sites of cholesterol crosslinks in peptides were refined by targeted MS/MS. Differential sites of crosslinking as a function of cholesterol concentration in both states were identified, with crosslinks found primarily in transmembrane helices and extramembranous loops of GlyR. Specific concentration-dependent sites of interaction between cholesterol and GlyR in both states 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. Differential cholesterol crosslinking patterns between resting and desensitized states were observed, highlighting state-dependent differences in GlyR lipid accessibility.