The Function and Surface Expression of the Organic Cation Transporter 1 (OCT1) is Affected by Free Cholesterol

Abstract

BackgroundThe function of the major renal organic cation transporter OCT2, is affected by plasma membrane cholesterol. OCT1, its liver counterpart, is important for the uptake of cationic drugs such as metformin into human hepatocytes. Fatty liver disease where free cholesterol accumulates in the liver can be a consequence of type 2 diabetes, which is treated with metformin. Thus, understanding how altered membrane cholesterol content impacts the function of OCT1 is important for the disposition of the drugs it transports.AimWe wanted to investigate to what extent the addition and the removal of free cholesterol from the plasma membrane would alter the function and expression of OCT1.MethodsHEK293 Flp‐In cells stably expressing OCT1 were used for all experiments. To add cholesterol, cells were treated with a cholesterol‐MCD complex in serum‐free media for 30 minutes. To remove cholesterol from the membrane cells were incubated for 30 minutes in serum‐free media containing methyl‐b‐cyclodextrin (MCD). Uptake of the radiolabeled model substrate MPP+ (1‐methyl‐4‐phenylpyridinium) was measured at 37°C. Michaelis‐Menten kinetics were determined under initial linear rate conditions (15 second uptake). OCT1 expression at the plasma membrane was determined using a surface biotinylation assay.ResultsTreatment of the cells with increasing concentrations of both cholesterol and MCD resulted in a concentration‐dependent decrease of OCT1 transport. Kinetics of OCT1‐mediated MPP+ uptake following cholesterol treatment demonstrated that the Km values increased from 35 ± 6.7 μM to 73 ± 10.2 μM while the Vmax values were essentially unchanged: 7.5 ± 0.6 pmol/mg x min and 8.4 ± 0.3 pmol/mg x min, respectively. As a consequence, the capacity of OCT1 decreased by 45%. Treatment of cells with MCD slightly decreased the Km value to 26 ± 4.9 μM while the Vmax value stayed the same at 7.7 ± 0.5 pmol/mg x min. With respect to surface expression, treatment with 20 mM MCD treatment decreased surface expression of OCT1 by about 65% while cholesterol treatment resulted in a 30% decrease.Summary and ConclusionBoth the addition and removal of free cholesterol from the plasma membrane impacts OCT1 transport and expression at the plasma membrane. These results suggest that drug disposition will be different in patients with either higher or lower free cholesterol in the liver, which can occur in fatty liver disease or type 2 diabetes.