TY - JOUR T1 - Plasma membrane cholesterol regulates the allosteric binding of 1-methyl-4-phenylpyridinium (MPP+) to organic cation transporter 2 (OCT2, SLC22A2) JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther DO - 10.1124/jpet.119.260877 SP - jpet.119.260877 AU - Severin Hoermann AU - Zhibo Gai AU - Gerd A Kullak-Ublick AU - Michele Visentin Y1 - 2019/01/01 UR - http://jpet.aspetjournals.org/content/early/2019/10/17/jpet.119.260877.abstract N2 - The human organic cation transporter 2 (OCT2) mediates the first step of tubular secretion of most positively charged substances. The present work describes the role of plasma membrane cholesterol in OCT2 activity. Human embryonic kidney 293 cells overexpressing OCT2 (OCT2-HEK293) and wild-type HEK293 cells (WT-HEK293) were employed. Cellular cholesterol content, assessed by thin layer chromatography, was manipulated using empty methyl-β-cyclodextrin (mβcd) and cholesterol-presaturated mβcd (RAMEB). The effect of mβcd on OCT2 protein stability and oligomerization state was evaluated by immunofluorescence and western blotting. Transport activity of OCT2 was measured using [3H]1-methyl-4-phenylpyridinium (MPP+). A 20-minute incubation with mβcd reduced total cellular cholesterol content by 40-60% as compared with that in untreated cells, without altering the content of the other main lipid species. In this condition, OCT2-mediated uptake of MPP+ was reduced by ≈ 50%. When cells were co-incubated with empty mβcd and cholesterol saturated mβcd (RAMEB), cholesterol content as well as OCT2-mediated uptake of MPP+ were comparable to those in untreated cells, suggesting that the mβcd effect on OCT2 activity was cholesterol-dependent. In untreated cells, the MPP+ influx kinetics was allosteric, whereas in cells treated with mβcd, one binding site was observed. Our findings suggest that changes in cellular cholesterol content can dramatically alter OCT2-mediated transport, potentially resulting in abnormal tubular secretion and unexpected drug toxicity and drug-drug interactions.SIGNIFICANCE STATEMENT Plasma membrane cholesterol is important for the allosteric properties of OCT2. From a pharmacological standpoint, the variability in cholesterol content stemming from certain pathophysiological conditions such as aging and acute kidney injury should be taken into account as additional source of interpatient pharmacokinetics/pharmacodynamics variability and unexpected toxicity profile of OCT2 substrates, which can escape the preclinical and clinical development. ER -