Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry
biorxiv(2020)
摘要
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 lipid-accessible 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.
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关键词
Human serotonin transporter,crosslinking mass spectrometry,tandem mass spectrometry,azi-cholesterol
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