Vol. 286, Issue 1, 354-361, July 1998

Functional Characterization of an Organic Cation Transporter (hOCT1) in a Transiently Transfected Human Cell Line (HeLa)¹

Lei Zhang², Marci E. Schaner and Kathleen M. Giacomini

Department of Biopharmaceutical Sciences, University of California, San Francisco, San Francisco, California

Recently, a polyspecific organic cation transporter, hOCT1, was cloned from human liver. To date, limited studies examining the functional characteristics of the transporter have been performed. The purpose of the present study was to develop a mammalian expression system for hOCT1 and to characterize the interactions of various compounds with the cloned transporter. Lipofection was used to transiently transfect the hOCT1 plasmid DNA in a human cell line, HeLa. We tested the interaction of an array of organic cations and other compounds with hOCT1 by determining K_i values in inhibiting C-tetraethylammonium (TEA) transport in the transfected cells. Transient expression of hOCT1 activity was observed between 24 and 72 hr post-transfection, with maximal expression at approximately 40 hr. TEA transport was temperature dependent and saturable with V_max and K_m values of 2.89 ± 0.448 nmol/mg protein/30 min and 229 ± 78.4 µM, respectively. ¹⁴C-TEA uptake in hOCT1 plasmid DNA-transfected HeLa cells was trans-stimulated by unlabeled TEA and 1-methyl-4-phenyl-pyridinium. Organic cations, including clonidine, quinine, quinidine and verapamil (0.1 mM), significantly inhibited ¹⁴C-TEA uptake, whereas the organic anion, p-aminohippuric acid (5 mM), did not. The neutral compounds, corticosterone (K_i, 7.0 µM) and midazolam (K_i, 3.7 µM) potently inhibited ¹⁴C-TEA uptake. The K_i values of several compounds in interacting with hOCT1 differed substantially from the corresponding values for the rat organic cation transporter, rOCT1, and the human kidney-specific organic cation transporter, hOCT2, determined in previous studies. Transiently transfected HeLa cells represent a useful tool in studying the interactions and kinetics of organic cations and other xenobiotics with hOCT1 and in understanding the molecular events involved in organic cation transport.