Phenylalanine 368 of multidrug resistance-associated protein 4 (MRP4/ABCC4) plays a crucial role in substrate-specific transport activity

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• 作者：Hanneke G.M. Wittgen^a ; ¹ ; Jeroen J.M.W. van den Heuvel^a ; ¹ ; Elmar Krieger^b ; Gijs Schaftenaar^b ; Frans G.M. Russel^a ; Jan B. Koenderink^a ; ^{J.Koenderink@pharmtox.umcn.nl}
• 关键词：MRP ; multidrug resistance-associated protein ; eYFP ; enhanced yellow fluorescent protein ; HEK293 ; human embryonic kidney 293 cells ; DMEM ; Dulbecco Eagle's modified medium ; F368/Phe368 ; phenylalanine at position 368 of MRP4 ; W995/Trp995 ; tryptophan at posit
• 刊名：Biochemical Pharmacology
• 出版年：2012
• 期刊代码：2_00062952
• 类别：pha
• 出版时间：1 August, 2012
• 卷：84
• 期：3
• 页码：366-373
• 文件大小：1215 K

摘要

Multidrug resistance-associated protein 4 (MRP4) is a membrane transporter that mediates the cellular efflux of a wide range of anionic drugs and endogenous molecules. MRP4 transport can influence the pharmacokinetics of drugs and their metabolites, therefore more knowledge about the molecular determinants important for its transport function would be of relevance. Here, we substituted amino acids Phe³⁶⁸, Trp⁹⁹⁵, and Arg⁹⁹⁸ with conservative or non-conservative residues, and determined the effect on transport of the model substrates estradiol 17-尾-d-glucuronide (E₂17尾G), cyclic guanosine monophosphate (cGMP), methotrexate (MTX), and folic acid into membrane vesicles isolated from baculovirus transduced HEK293 cells overexpressing the mutant MRP4 proteins. This revealed that all Arg⁹⁹⁸ mutations appeared to be deleterious, whereas the effect of a Phe³⁶⁸ or Trp⁹⁹⁵ replacement was dependent on the amino acid introduced and the substrate studied. Substitution of Phe³⁶⁸ with Trp (F368W) induced a gain-of-function of E₂17尾G transport and a loss-of-function of MTX transport, which could not be attributed to an altered substrate binding. Moreover, we did not observe any modification in ATP or ADP handling for F368W. These results, in combination with docking of substrates in a homology model of MRP4 in the inward- and outward-facing conformation, suggest that Phe³⁶⁸ and Trp⁹⁹⁵ do not play an important role in the initial binding of substrates. They, however, might interact with the substrates during rearrangement of helixes for substrate translocation, funneling the substrates to the exit site in the outward-facing conformation.