Human Multidrug and Toxin Extrusion Protein 1: Symmetry of substrate fluxes
dc.contributor.advisor | Wright, Stephen H. | en_US |
dc.contributor.author | Dangprapai, Yodying | |
dc.creator | Dangprapai, Yodying | en_US |
dc.date.accessioned | 2011-10-14T21:51:57Z | |
dc.date.available | 2011-10-14T21:51:57Z | |
dc.date.issued | 2011 | |
dc.identifier.uri | http://hdl.handle.net/10150/145435 | |
dc.description.abstract | Human multidrug and toxin extrusion 1 (hMATE1) is a major candidate for being the molecular identity of organic cation/proton (OC/H+) exchange activity in the luminal membrane of renal proximal tubules (RPT). Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, I measured the time course of hMATE1-mediated efflux of the prototypic MATE1-substrate, [3H]1-methyl-4-phenylpyridinium ([3H]MPP), under a variety of conditions, including different values for intra- and extracellular pH, from CHO cells that stably expressed hMATE1. I showed that an IC50/Ki for interaction between extracellular H+ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] were not significantly different (P = 0.6). To test a hypothesis that H+ interacts symmetrically with each face of hMATE1, kinetics of interaction between intracellular H+ and inward-facing hMATE1 were determined using the efflux protocol. The IC50 for interaction with H+ was 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1. The efflux protocols demonstrated in this study are a potential means to examine kinetics at cytoplasmic face of hMATE1 and also a practical tool to screen uptake of substrates at extracellular face of hMATE1. | |
dc.language.iso | en | en_US |
dc.publisher | The University of Arizona. | en_US |
dc.rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. | en_US |
dc.subject | hMATE1 | en_US |
dc.subject | organic cation | en_US |
dc.subject | pH | en_US |
dc.subject | renal proximal tubule | en_US |
dc.title | Human Multidrug and Toxin Extrusion Protein 1: Symmetry of substrate fluxes | en_US |
dc.type | Electronic Dissertation | en_US |
dc.type | text | en_US |
dc.identifier.oclc | 752261494 | |
thesis.degree.grantor | University of Arizona | en_US |
thesis.degree.level | doctoral | en_US |
dc.contributor.committeemember | Dantzler, William H. | en_US |
dc.contributor.committeemember | Delamere, Nicholas A. | en_US |
dc.contributor.committeemember | Brooks, Heddwen L. | en_US |
dc.identifier.proquest | 11648 | |
thesis.degree.discipline | Graduate College | en_US |
thesis.degree.discipline | Physiological Sciences | en_US |
thesis.degree.name | Ph.D. | en_US |
refterms.dateFOA | 2018-06-11T14:08:36Z | |
html.description.abstract | Human multidrug and toxin extrusion 1 (hMATE1) is a major candidate for being the molecular identity of organic cation/proton (OC/H+) exchange activity in the luminal membrane of renal proximal tubules (RPT). Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, I measured the time course of hMATE1-mediated efflux of the prototypic MATE1-substrate, [3H]1-methyl-4-phenylpyridinium ([3H]MPP), under a variety of conditions, including different values for intra- and extracellular pH, from CHO cells that stably expressed hMATE1. I showed that an IC50/Ki for interaction between extracellular H+ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] were not significantly different (P = 0.6). To test a hypothesis that H+ interacts symmetrically with each face of hMATE1, kinetics of interaction between intracellular H+ and inward-facing hMATE1 were determined using the efflux protocol. The IC50 for interaction with H+ was 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1. The efflux protocols demonstrated in this study are a potential means to examine kinetics at cytoplasmic face of hMATE1 and also a practical tool to screen uptake of substrates at extracellular face of hMATE1. |