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Influence of [H + ] on MPP efflux from CHO cells expressing hMATE1
Author(s) -
Dangprapai Yodying,
Wright Stephen H
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.1024.11
Subject(s) - efflux , extracellular , chemistry , washout , cytoplasm , biophysics , kinetics , ion transporter , membrane transport , transporter , substrate (aquarium) , membrane , chromatography , biochemistry , biology , ecology , oceanography , physics , quantum mechanics , gene , geology
The human multidrug and toxin extrusion protein, hMATE1, is a key transporter mediating organic cation (OC)/hydrogen ion (H + ) exchange at the luminal membrane of renal proximal tubule (RPT) cells. Physiologically, hMATE1 secretes OCs from RPT cells into the filtrate. However, kinetic studies of hMATE1 have largely been confined to measurement of OC uptake into hMATE1‐expressing cells. To characterize transport activity of hMATE1 in its native mode, we developed a method to study efflux of 1‐methy‐4‐phenylpyrinidium (MPP) in CHO cells expressing hMATE1. After loading cells with [ 3 H]MPP in pH 8.5 buffer, time course of washout was determined as a function of [H + ] in the washout buffer. Efflux involved at least two phases, a fast phase representing ~87% of the accumulated MPP, and a very slow phase that could be subtracted from the first for analysis. Rate constant for MPP efflux of the fast phase (presumably from the cytoplasm) increased significantly (p < 0.0001) when external [H + ] was raised from 3.16 nM (pH 8.5; K=0.22 min −1 ) to 40 nM (pH 7.4; K=0.51 min −1 ). Half‐maximal MPP efflux occurred at an extracellular [H + ] of 4.42 nM (pH 8.35), a concentration not significantly different from the IC 50 for inhibition of MPP uptake produced by extracellular [H + ] (7.26 nM; pH 8.14). This method offers the promise of measuring the kinetics and selectivity of substrate interaction at the cytoplasmic face of hMATE1. (NIH R56DK080801)