Transport Turnover Rates for Human OCT2 and MATE1 Expressed in Chinese Hamster Ovary Cells

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).

Determination of metal ion transport rate of human ZIP4 using stable zinc isotopes

“…These results allowed us to estimate the apparent turnover number of hZIP4 to be 0.08±0.02 s -1 (mean±standard error, S.E., n=3). This value is generally consistent with the turnover numbers of representative carrier proteins (66) and close to a panel of slow carriers, including VMAT1 (0.2 s -1 ) (67), MATE1 (0.4 s -1 ) (68), NET (0.11 s -1 ) (69), Tyt1 (0.4 s -1 ) (70), and Gltph (0.14 s -1 ) (71). The slow transport rate and the saturable kinetics both suggest that hZIP4 is best characterized as a carrier.…”

“…Internal standardization was accomplished inline using the ISIS valve and a 200 ng/g internal standard solution in 3% (v/v) trace nitric acid in ultrapure water consisting of Bi, In, 6 Li, Sc, Tb, and Y (IV-ICPMS-71D, Inorganic Ventures, Christiansburg, VA, USA). The isotopes selected for analysis were 31 P, 64 Zn, 66 Zn, 67 Zn, 68 Zn, 70 Zn and 6 Li, 45 Sc, and 89 Y for internal standardization. Continuing calibration blanks (CCBs) were run every 10 samples and a continuing calibration verification standard was analyzed at the end of every run for a 90-110% recovery.…”

Anion Pathways in the NarK Nitrate/Nitrite Exchanger

“…(ii) The steering speed of 0.1 Å/ns, which originally seemed to be reasonably slow, might still be too fast to allow full relaxation of the protein. To our knowledge, there are no published experimental turnover rates of NarK; but most transporters are in the range of several to several hundred per second. , Therefore, the anion migration through the pore may be on the time scale of milliseconds, which is currently beyond our reach. (iii) We have only simulated the step of individual anions passing through the pore, and it is possible that large-scale conformational changes for NO 2 – transport are involved in other places or steps that are not covered in the present study.…”

Exploring Amino Acid Transporters as Therapeutic Targets for Cancer: An Examination of Inhibitor Structures, Selectivity Issues, and Discovery Approaches

“…The movement of a substrate across a membrane, where the transporter moves from the outward open to the inward open conformation, is called a translocation cycle. The transporter turnover rate is the number of molecules moved by a single transporter per second [ 27 ]. Substrates are compounds that bind to and are translocated by the solute carrier.…”