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 percent has been shown to have negligible chemical activity, suggesting that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold. 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, a photoactivatable nonspecific cross-linker. After photoactivation, cross-linked cholesterol-GlyR was trypsinized and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS), and sites of direct covalent attachment to peptides were refined by targeted MS/MS. Differential patterns of dozens of cholesterol-GlyR cross-links were identified in these comparative studies, with sites of cross-linking found primarily in the fourth transmembrane helix and extramembranous connecting loops and mapping the lipid-accessible surface of the receptor. Unique cross-linking observed in both reduced and elevated cholesterol composition suggests different apo-state structural conformations of GlyR as a function of cholesterol concentration and, in the latter studies, identified potential specific binding sites for cholesterol in the receptor.

Altered serotonin (5-HT) levels contribute to disease states such as depression and anxiety. Synaptic serotonin concentration is partially regulated by the serotonin transporter (SERT), making this transporter an important therapeutic target. This study seeks to examine the lipid accessible domains of hSERT to provide critical information regarding the apo-state of this transporter in a lipid environment. Recombinant hSERT was inducibly expressed in a human cell line. Solubilized SERT was purified by affinity chromatography using a FLAG Tag and reconstituted into mixed lipid vesicles containing our photoactivatable lipid probe. The lipidaccessible domains of the reconstituted transporter in membranes in its apo-state were probed via photocrosslinking to azi-cholesterol followed by quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LC-MS). MS studies identified crosslinks in three transmembrane loops consistent with the known topology of SERT. Surprisingly, the amino-and carboxy-terminal domains were similarly crosslinked by cholesterol, suggesting that these regions may also be intimately associated with the lipid bilayer. The data presented herein assist in further refining our understanding of the topography of the apo-state of hSERT via analysis of lipid accessibility. INTRODUCTION:

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AbstractPentameric 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.SignificanceIon 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.

Tau-microtubule ordering (in an active system) in the absence of paclitaxel and, in particular, the effect of biological cations on microtubule self-assembly using transmission electron microscopy (TEM) and synchrotron small-angle X-ray scattering (SAXS).