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J. Biol. Chem., Vol. 280, Issue 26, 24481-24490, July 1, 2005

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Alkali Cation Binding and Permeation in the Rat Organic Cation Transporter rOCT2^*

Bernhard M. Schmitt and Hermann Koepsell

From the Department of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany

Organic cation transporters of the OCT family mediate downhill transport of organic cations, compatible with carrier, pore, or gate-lumen-gate mechanisms. We studied rat OCT2 expressed in Xenopus oocytes by the two-electrode voltage-clamp technique, including membrane capacitance (C_m) monitoring. Choline, a transported cationic substrate, elicited the expected inward currents but also elicited decreases of C_m. Similar C_m decreases were caused by the non-transported inhibitors tetrabutylammonium (a cation) and corticosterone (uncharged). Effects on C_m were voltage-dependent, with a maximum at –140 mV. These findings suggest that the empty rOCT2 protein can undergo an electrogenic conformation change, with one conformation highly favored at physiological voltage. Moreover, alkali cations elicited considerable inward currents and inhibited uptake of [¹⁴C]tetraethylammonium with a sequence Cs⁺ > Rb⁺ > K⁺ > Na⁺ Li⁺. Cs⁺ affected current and capacitance with similar affinity (K_0.5 50 mM). Tetraethylammonium inhibited Cs⁺ currents in a concentration-dependent manner. Conversely, Cs⁺ inhibited tetraethylammonium uptake by a competitive mechanism. Activation energy of the currents estimated from measurements between 12 °C and 32 °C was 81 kJ/mol for Cs⁺ and 39 kJ/mol for tetramethylammonium, compatible with permeation of Cs⁺ through rOCT2 along the same path as organic substrates and by a mechanism different from simple electrodiffusion. Rationalization of Cs⁺ selectivity in terms of a pore pointed to a pore diameter of 4 Å. Intriguingly, that value matches the known selectivity of rOCT2 for organic compounds. Our data show that selective permeability of rOCT2 is not determined by ligand affinity but might rather be understood in terms of the ion channel concept of a distinct "selectivity filter."

Received for publication, December 27, 2004 , and in revised form, May 4, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-931-312701; Fax: 49-931-312087; E-mail: bernhard.schmitt{at}mail.uni-wuerzburg.de.

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