Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa phosphoglycoprotein that mediates the efflux of structurally diverse endo- and xenobiotics across biological membranes, and is known to play roles in drug disposition and resistance. The goal of the present study was to examine the functional importance of the region proximal to transmembrane helix 17 (TM17) of MRP1 by mutational analysis of seven conserved amino acids in this region. Thus, Glu1253, Glu1255, Val1261, Glu1262, Arg1263, Glu1266, and Tyr1267 were initially replaced by Ala, and after expression in HEK293T cells, the properties of the mutant proteins were investigated. All of the mutant proteins were expressed at levels comparable to wild-type MRP1, indicating that these residues are not critical for MRP1 biosynthesis. Vesicular transport assays showed that Ala-substitution of Glu1253 and Glu1262 significantly reduced 17β-estradiol 17-(β-D-glucuronide) (E217βG) and leukotriene C4 (LTC4) transport by 30-75% (p < 0.05), while Ala-substitution of Glu1255 and Glu1266 had no effect. Transport activity of the same-charge mutant E1253D was comparable to wild-type MRP1, while transport by E1262D remained reduced (by 50-75%) (p < 0.05). Kinetic analysis suggests that E1253A and E1262A exhibit reduced E217βG uptake as a result of a decreased uptake affinity (Km), while the reduced transport of E1262D was associated with a reduction in Vmax. Reciprocal mutations of potential interhelical bonding partners of Glu1253 and Glu1262 (Lys1141 and Arg1142, respectively), identified by examination of an atomic homology model of MRP1, did not significantly enhance MRP1 function. This suggests that even if bonding interactions exist between the side-chains of these two pairs of amino acids, the interactions are not exclusive. These findings also suggest that Glu1253 and Glu1262 have unique and complex roles in substrate binding and/or translocation. Ala-substitution of Val1261, Arg1263 and Tyr1267 caused a small reduction in E217βG transport (by 25-35%) (p < 0.05), while reductions in LTC4 transport were somewhat more substantial (by 30-55%) (p < 0.05). In conclusion, these studies have provided the first evidence of the functional importance of anionic residues in the COOH-proximal region of TM17 of MRP1. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-09-29 16:54:45.378
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/5560 |
| Date | 26 April 2010 |
| Creators | MOLINSKI, STEVEN |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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