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

The driving forces mediating tetraethylammonium (TEA) transport were system atically assessed in Xenopus oocytes and MDCK cells expressing organic cati on transporter (OCT) 2 cloned from rat kidney (rOCT2). In rOCT2 cRNA-inject ed oocytes, uptake of [C-14]TEA was saturable, with an estimated Michaelis constant (K-m) of 393 mu M, and was specifically inhibited by organic catio ns. Furthermore, TEA uptake demonstrated two distinct components, one that was potential sensitive and one that was pH sensitive. When membrane potent ial 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 vali nomycin), decreasing external medium pH significantly reduced TEA uptake. C onsistent with the potential sensitivity of uptake, electrophysiological an alysis of rOCT2-injected oocytes demonstrated movement of positive charge i nto 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-rO CT2) showed mediated, potential-sensitive, pH-sensitive TEA uptake (K-m = 4 8 mu M). TEA efflux from preloaded MDCK-rOCT2 cells was stimulated by exter nally applied (trans) tetramethylammonium but was trans-inhibited by H+ (ex ternal pH 5.4). The effect of external H+ was to modulate rOCT2-mediated tr ansport. Thus rOCT2 is a potential-driven transporter, not an organic catio n/H+ exchanger, consistent with a physiological role in the basolateral ent ry step in renal organic cation secretion.