Organic cation transporters are important for the elimination of many drugs and toxins from the body. In the present study, substrate-transporter interactions were investigated in Chinese hamster ovary cells stably transfected with either the human or rabbit orthologs of the principal organic cation transporter in the kidney, OCT2. IC 50 values, ranging from 0.04 M to Ͼ3 mM, for inhibition of [ 14 C]tetraethylammonium transport were determined for more than 30 structurally diverse compounds. Although the two OCT orthologs displayed similar IC 50 values for some of these compounds, the majority varied by as much as 20-fold. Marked differences in substrate affinity were also noted when comparing hOCT2 to the closely related homolog hOCT1. These data suggest the molecular determinants of substrate binding differ markedly among both homologous and orthologous OCT transporters. The software package Cerius 2 (Accelrys, San Diego, CA) was used to generate a descriptorbased, two-dimensional, quantitative structure-activity relationship (QSAR) to produce a model relating the affinity of hOCT2 to particular physicochemical features of substrate/ inhibitor molecules (r 2 ϭ 0.81). Comparative molecular field analysis (Tripos, St. Louis, MO) was used to generate threedimensional QSARs describing the structural basis of substrate binding to hOCT2 and rbOCT2 (q 2 ϭ 0.60 and 0.53, respectively, and each with r 2 ϭ 0.97). The quality of the models was assessed by their ability to successfully predict the inhibition of a set of test compounds. The current models enabled prediction of OCT2 affinity and may prove useful in the prediction of unwanted drug interactions at the level of the renal secretory process.The kidneys play a key role in the secretion and subsequent elimination of drugs, toxins, and other xenobiotics from the body (Koepsell et al

Pelis, Ryan M., Wendy M. Suhre, and Stephen H. Wright. Functional influence of N-glycosylation in OCT2-mediated tetraethylammonium transport. Am J Physiol Renal Physiol 290: F1118 -F1126, 2006. First published December 20, 2005 doi:10.1152/ajprenal.00462.2005.-OCT2, an organic cation transporter critical for removal of many drugs and toxins from the body, contains consensus sites for N-glycosylation at amino acid position 71, 96, and 112. However, the extent to which these sites are glycosylated by the cell, and the influence glycosylation has on OCT2 function, remains unknown. To address these issues, the acquisition of N-glycosylation was disrupted by mutating the amino acid asparagine (N) to glutamine (Q) at these sites in the rabbit ortholog of OCT2, which was expressed in Chinese hamster ovary cells. Disruption of N-glycosylation followed by Western blotting indicated that each site is indeed glycosylated and that OCT2 contains no other sites of Nglycosylation. Plasma membrane expression (determined by surface biotinylation) of the N112Q mutant, but not N71Q or N96Q mutants, was fourfold lower than that of wild-type OCT2, and unglycosylated OCT2 (N71Q/N96Q/N112Q) was sequestered in an unidentified intracellular compartment. The N71Q, N96Q, and N112Q mutants had a higher affinity (ϳ2-fold) for tetraethylammonium (TEA). Maximum transport rate was reduced in the N96Q (3-fold) and N112Q (5-fold) mutants, but not the N71Q mutant, and unglycosylated OCT2 failed to transport TEA (associated with its absence in the plasma membrane). Whereas the reduction in maximum transport rate of the N112Q mutant is consistent with its reduced plasma membrane expression, the lower rate of the N96Q mutant, which appeared to traffic properly, suggests that glycosylation at N96 increases the transporter turnover number. SLC22A2; organic cation; rabbit ORGANIC CATION TRANSPORTERS (OCTs) in the SLC22A gene family transport a variety of organic cations (OCs), including clinically important therapeutics (e.g., cimetidine) and environmental toxins (e.g., nicotine). Three OCTs (OCT1, OCT2, and OCT3) have been cloned, and each appears to be restricted to the basolateral membrane of barrier epithelia (32). In the renal proximal tubule, an important site for controlling plasma levels of OCs, all three OCTs are expressed and provide an essential pathway for OC uptake, the first step in tubular secretion (32). Depending on the prevailing set of electrical and chemical gradients, OCT-mediated transport can occur either via electrogenic facilitated diffusion (i.e., driven by the negative membrane potential) or OC/OC exchange (4, 5, 13). In humans, OCT2 (SLC22A2) has been postulated to be the greatest contributor to tubular OC secretion since its expression (as indicated by mRNA and protein levels) predominates over that of OCT1 and OCT3 (19). However, both OCT1 and OCT3 may play significant roles in the renal handling of select OCs (e.g., those transported inefficiently by OCT2) (32, 34). OCT1, 2, and 3 belong to a larger family of solute carr...