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State-dependent mapping of GlyR-cholesterol interactions by coupling crosslinking with mass spectrometry

Pentameric ligand-gated ion channel (pLGIC) allostery is dependent on dynamic associations with its diverse environment. The cellular membrane’s lipid composition influences channel function with cholesterol being a key regulator of channel activity. Human α1 glycine receptor (GlyR) was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at physiological cholesterol:lipid ratios with aliquots of azi-cholesterol, a photoactivatable non-specific crosslinker. The receptor in vesicles was then enriched in either a resting, open, or desensitized state prior to photocrosslinking. Following photoactivation, crosslinked cholesterol-GlyR was trypsinized and sites of direct covalent attachment to peptides were identified by targeted MS/MS. Dozens of state-dependent crosslinks were identified and differential patterns of cholesterol-GlyR crosslinks were observed in the extracellular region nearing the lipid bilayer, in the M4 transmembrane helix, and in the large intracellular M3-M4 loop. Unique crosslinks in comparative studies identify changes in lipid accessibility or modulation of hydrophobic cavities in GlyR as a function of receptor allostery. Most notably, the outward twisting of M4 and differential crosslinking within the M3-M4 loop provide new insight into allosteric repositioning of GlyR. More generally, this study provides an accurate and sensitive approach to mapping the protein-lipid interactions to discern state-dependent structural movements of membrane proteins embedded in lipid-bilayers. Significance Ion channels are highly allosteric molecular machines whose structure and function are sensitive to lipids and ligands. While the structures of many pLGICs are known, these are often truncated forms of the receptor in a membrane-mimetic environment locked in ligand-bound conformational states that may not accurately reflect the conformation and dynamics of the receptor in a native lipid environment. Crosslinking coupled with mass spectrometry (CX-MS) has the capability of interrogating the structure of full-length receptors in a lipid environment. In this study, CX-MS was used to identify state-dependent cholesterol-GlyR interactions to identify differential cholesterol accessibility as a function of channel dynamics upon gating and desensitization.