Differential State-Dependent Crosslinking of Azi-Cholesterol with Human A1 Glycine Receptor Using Mass Spectrometry

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. Human a1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol:lipid ratios above the cholesterol activity threshold with aliquots of azi-cholesterol. State-dependent crosslinking studies of receptors primarily in its resting (no glycine), desensitized (10mM glycine) and open (F207A/A288G, 30nM ivermectin) states were performed. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsinized, mass fingerprinted by tandem mass spectrometry (MS), and sites of cholesterol crosslinks in peptides were refined by targeted MS/MS. Differential cholesterol crosslinking patterns between resting, desensitized, and open states were observed, highlighting state-dependent differences in GlyR lipid accessibility. Distinct state-dependent crosslinking patterns indicative of alterations in either the lipid environment and/or channel structure were observed throughout GlyR, most prominently observed in the M4 transmembrane helix, ECD loops and regions nearing the bilayer interface, and the large intracellular M3-M4 loop. Strikingly, crosslinking patterns within the M3-M4 loop offer insight into the generalized structure of this unresolved region of all current pLGIC structural models, by suggesting the crosslinked regions of this intracellular loop are intimately associated or buried within the lipid bilayer. Taken together, crosslinking coupled with tandem MS has the capability to accurately probe and define physiological protein frameworks which can aid in the refinement of allosteric modulation and current structural models.