TABLE 3.

Transport kinetics of zebrafish Oatp1d1 after disruption of four potential N-glycosylation sites (N122Q, N133Q, N499Q, and N512Q) and simultaneous mutation of all four asparagine residues (Asn-122/133/499/512)

Transport measurements were performed 24 h after transient transfection in HEK293 cells. Oatp1d1-overexpressing HEK293 cells were preincubated in the transport medium (145 mm NaCl, 3 mm KCl, 1 mm CaCl₂, 0.5 mm MgCl₂, 5 mm d-glucose, and 5 mm HEPES, pH 7.4) for 10 min at 37 °C. To assess transport, medium was removed, and the same medium containing the substrate was added. Concentration dependence of LY uptake is expressed as transport rate (nmol/mg protein/min) over LY concentration (μm) after a 15-min incubation with LY of varying concentrations. The uptake into vector-transfected HEK293 cells (mock cells) was subtracted to obtain transporter-specific uptake, and data were fitted in the GraphPad Prism version 5. Each value represents the mean ± S.E. from triplicate determinations of representative experiments (each experiment was repeated three times).

	Km (LY)	c.i.	Vm (LY)	c.i.
Oatp1d1 WT	28.3	23.9–32.6	127	119–135
N122Q	23.8	17.3–30.3	59.5	53.3–65.7
N133Q	24.8	19.2–30.4	76.9	70.1–83.7
N499Q	44.7	36.8–52.7	85.2	80.2–90.1
N512Q	44.7	30.7–58.7	111	99.2–122
N122Q/N133Q/N499Q	26.5	17.3–30.3	62.7	53.3–70.7
N122Q/N133Q/N499Q/N512Q	21.3	13.9–28.6	18.8	16.2–20.7