Functional influence ofN-glycosylation in OCT2-mediated tetraethylammonium transport

Abstract

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...

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…OCT2 contains consensus sites for N-glycosylation at amino acid positions 71, 96, and 112 (Pelis et al, 2006). Mutation of these asparagine residues demonstrates that each site is indeed glycosylated (Pelis et al, 2006).…”

“…Substitution at amino acids 96 and 112, but not 71, reduces the transport rate of OCT2. The reduced transport rate of the 112 50 mutant OCT2 is due to insufficient plasma membrane expression and intracellular retention (Pelis et al, 2006). The mutant protein containing a substituted amino acid at position 96 traffics properly to the cell membrane, suggesting that glycosylation of this residue reduces the rate of transport by increasing transporter turnover.…”

Characterization of the Disposition and Toxicokinetics ofN-Butylpyridinium Chloride in Male F-344 Rats and Female B6C3F1 Mice and Its Transport by Organic Cation Transporter 2

“…To determine the transport of TEA or NBuPy-Cl by human organic cation transporter 2 (hOCT2), Chinese hamster ovary ( CHO cells and CHO_hOCT2 cells were maintained in Ham's F12 medium with fetal bovine serum (10%, v/v), penicillin (100 unit/ml), streptomycin (100 g/ml), and hygromycin B (100 g/ml, only for CHO_hOCT2 cells) (Pelis et al, 2006). For the transport assay, cells in 12-well cell culture plates (550,000 cells/ml/well) were maintained in the incubator at 37°C for 24 h. The medium was then aspirated carefully before the experiment, and the cells were immediately rinsed twice with 1 ml of Waymouth's buffer (WB, in mM: 135 NaCl, 13 HEPES-NaOH, 28 D-glucose, 5 KCl, 1.2 MgCl 2 , 2.5 CaCl 2 , and 0.8 MgSO 4 , pH 7.4) at room temperature.…”

Influence of Estrogen and Xenoestrogens on Basolateral Uptake of Tetraethylammonium by Opossum Kidney Cells in Culture

“…Crude membranes were prepared from OK cells using a modification of the method described by our laboratory (Pelis et al, 2006). OK cells were rinsed twice with PBS and the confluent monolayer was scraped from either one 44-cm 2 or six separate 4.7-cm 2 permeable supports using a cell scraper.…”

“…Detection of immunoreactivity was performed using the West Femto Enhanced Chemiluminescence Detection system (Pierce Chemical). Relative immunoreactivity was determined using Image J 1.34 s Analysis Software (National Institutes of Health, Bethesda, MD) as described previously (Pelis et al, 2006).…”