Functional influence of N-glycosylation in OCT2-mediated tetraethylammonium transport.
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 N-glycosylation. 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 ( approximately 2-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.
Pelis
Ryan M
RM
Dept. of Physiology, College of Medicine, Univ. of Arizona, Tucson, AZ 85724, USA. rpelis@email.arizona.edu
Suhre
Wendy M
WM
Wright
Stephen H
SH
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D027701:Organic Cation Transport Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D000075102:Organic Cation Transporter 2; D026902:Potassium Channel Blockers / Q000493:pharmacokinetics*; D011817:Rabbits; D019789:Tetraethylammonium / Q000493:pharmacokinetics*
D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; D026902:Potassium Channel Blockers; C491013:SLC22A2 protein, human; D019789:Tetraethylammonium