期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 3, 页码 -出版社
MDPI
DOI: 10.3390/ijms23031472
关键词
transport; transporter turnover rate; organic cation; kidney
资金
- National Institutes of Health [1R01GM129777]
In this study, the turnover rates (TORs) of MATE1 and OCT2 proteins expressed in CHO cells were determined using a quantitative western blot method. The results showed that MATE1 and OCT2 proteins represented 25% and 24%, respectively, of the total expression on the cell surface. The calculated TOR values for MATE1 and OCT2 were consistent with values determined for other transporters.
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 J(max) 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).
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