Abstract
MATE1 (multidrug and toxin extruder 1) and OCT2 (organic cation transporter 2) play critical roles in organic cation excretion by the human kidney. The transporter turnover rate (TOR) is relevant to understanding both their transport mechanisms and interpreting the in vitro–in vivo extrapolation (IVIVE) required for physiologically-based pharmacokinetic (PBPK) modeling. Here, we use a quantitative western blot method to determine TORs for MATE1 and OCT2 proteins expressed in CHO cells. MATE1 and OCT2, each with a C-terminal V-5 epitope tag, were cell surface biotinylated and the amount of cell surface MATE1 and OCT2 protein was quantified by western analysis, using standard curves for the V5 epitope. Cell surface MATE1 and OCT2 protein represented 25% and 24%, respectively, of the total expression of these proteins in CHO cells. The number of cell surface transporters was ~55 fmol cm−2 for MATE1 and ~510 fmol cm−2 for OCT2. Dividing these values into the different Jmax values for transport of MPP, metformin, and atenolol mediated by MATE1 and OCT2 resulted in calculated TOR values (±SE, n = 4) of 84.0 ± 22.0 s−1 and 2.9 ± 0.6 s−1; metformin, 461.0 ± 121.0 s−1 and 12.6 ± 2.4 s−1; atenolol, 118.0 ± 31.0 s−1, respectively. These values are consistent with the TOR values determined for a variety of exchangers (NHEs), cotransporters (SGLTs, Lac permease), and uniporters (GLUTs, ENTs).
Highlights
The kidneys play the primary role in clearing the body of ‘organic cations’ (OCs) [1], including about 40% of prescribed drugs [2,3]
Renal OC secretion is a two-step process: the first is dominated by the electrogenic uniporter, organic cation transporter 2 (OCT2), which mediates basolateral OC uptake from the blood into renal proximal tubule (RPT) cells; the second is dominated by the MATE family transporter, MATE1
To determine the first of these parameters, we measured the kinetics of transport of several structurally distinct compounds (MPP, metformin, and, atenolol), acknowledging that different substrates can display different maximal rates of transport (e.g., [22]), into Chinese hamster ovary (CHO) cells that stably expressed either MATE1 or OCT2
Summary
The kidneys play the primary role in clearing the body of ‘organic cations’ (OCs) [1], including about 40% of prescribed drugs [2,3]. While this makes them clinically important pathways for drug clearance, it sets the stage for potentially deleterious drug–drug interactions [10,11,12] Working in concert, these transporters play a pivotal role in defining the pharmacokinetics (PK) of cationic drug elimination, are primary sites of drug–drug interactions (DDIs), and contribute to the nephrotoxicity of selected compounds [13,14]. These transporters play a pivotal role in defining the pharmacokinetics (PK) of cationic drug elimination, are primary sites of drug–drug interactions (DDIs), and contribute to the nephrotoxicity of selected compounds [13,14] Their activity is central to efforts to use physiologically-based pharmacokinetic (PBPK) models to describe drug clearance and predict potential DDIs and cellular toxicity in humans [15]. For MATE, our estimates of TORs were markedly larger (>1000-fold), reflecting substantial differences in the estimates of transporter expression and rates of transport
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