title: Quantitative Determination of Luminal and Abluminal Membrane Distributions of Transporters in Porcine Brain Capillaries by Plasma Membrane Fractionation and Quantitative Targeted Proteomics.
abstract: Abluminal or luminal localization of transporter in plasma membranes at the blood-brain barrier (BBB) is critical for their physiological and pharmacological roles. Therefore, the purpose of this study was to develop a new method to investigate membrane localization of transporters, through quantitative measurement of protein expression levels in fractionated plasma membrane prepared from porcine brain capillaries. Luminal-abluminal distribution ratios were calculated from the results of quantitative targeted absolute proteomics of fractionated membranes, after correction for cross-contamination based on measurements of luminal and abluminal membrane markers. BCRP expression was greater at the luminal membrane than at the abluminal membrane, supporting the usefulness of the distribution ratio as a quantitative indicator of localization. The distribution ratios suggested luminal-dominant localizations of GLUT1 and OATP3A1, and abluminal-dominant localizations of ABCA1 and FATP1. For OATP3A1, ABCA1 and FATP1, these results require reconsideration of their functions at the BBB. Species differences were examined using expression levels normalized to Na(+) /K(+) -ATPase. BCRP expression is dominant over multidrug resistance 1 expression in porcine BBB, as in other primates including humans. This methodology for quantitative measurement of protein localization is expected to improve our understanding of the roles of transporters at the BBB, and should be applicable to other polarized cells.
authors.affiliation: Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama, 930-0194, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kubo
authors.affiliation: Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
authors.forename: Sumio
authors.initials: S
authors.lastname: Ohtsuki
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D001812:Blood-Brain Barrier / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D002196:Capillaries / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D050612:Fatty Acid Transport Proteins / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D027361:Organic Anion Transporters / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine
chemical_list: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D050612:Fatty Acid Transport Proteins; D051272:Glucose Transporter Type 1; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D000254:Sodium-Potassium-Exchanging ATPase
keywords: blood-brain barrier; localization; mass spectrometry; membrane transport; pharmacoproteomics (PPx); polarity; quantitative targeted absolute proteomics (QTAP); species differences; transporter