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Am J Physiol Renal Physiol 277: F890-F898, 1999;
0363-6127/99 $5.00
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Vol. 277, Issue 6, F890-F898, December 1999

rOCT2 is a basolateral potential-driven carrier, not an organic cation/proton exchanger

Douglas H. Sweet and John B. Pritchard

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

The driving forces mediating tetraethylammonium (TEA) transport were systematically assessed in Xenopus oocytes and MDCK cells expressing organic cation transporter (OCT) 2 cloned from rat kidney (rOCT2). In rOCT2 cRNA-injected oocytes, uptake of [14C]TEA was saturable, with an estimated Michaelis constant (Km) of 393 µM, and was specifically inhibited by organic cations. Furthermore, TEA uptake demonstrated two distinct components, one that was potential sensitive and one that was pH sensitive. When membrane potential was intact, TEA uptake was largely unaffected by changes in medium pH; when the oocyte membrane was depolarized (K+ in = out = 102.5 mM, plus valinomycin), decreasing external medium pH significantly reduced TEA uptake. Consistent with the potential sensitivity of uptake, electrophysiological analysis of rOCT2-injected oocytes demonstrated movement of positive charge into the oocyte upon TEA addition. To further evaluate the nature of the pH effect and assess the properties of rOCT2 in a renal epithelium, rOCT2 was introduced into MDCK cells. A stably transfected single cell clone (MDCK-rOCT2) showed mediated, potential-sensitive, pH-sensitive TEA uptake (Km = 48 µM). TEA efflux from preloaded MDCK-rOCT2 cells was stimulated by externally applied (trans) tetramethylammonium but was trans-inhibited by H+ (external pH 5.4). The effect of external H+ was to modulate rOCT2-mediated transport. Thus rOCT2 is a potential-driven transporter, not an organic cation/H+ exchanger, consistent with a physiological role in the basolateral entry step in renal organic cation secretion.

rat; kidney; Xenopus laevis; expression cloning; Madin-Darby canine kidney; transfection


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