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J. Biol. Chem., Vol. 278, Issue 35, 32497-32500, August 29, 2003

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Transport Kinetics of Uncoupling Proteins

ANALYSIS OF UCP1 RECONSTITUTED IN PLANAR LIPID BILAYERS^*

Eva Urbánková , Anna Voltchenko , Peter Pohl ¶ ||, Petr Jeek  and Elena E. Pohl  **

From the Department of Membrane Transport Biophysics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic, Neuroscience Research Center, Humboldt-University, Berlin, Germany, ^¶Research Institute of Molecular Pharmacology, Berlin, and ^||Experimental Biophysics, Institute of Biology, Humboldt-University, Berlin

According to alternative hypotheses, mitochondrial uncoupling protein 1 (UCP1) is either a proton channel ("buffering model") or a fatty acid anion carrier ("fatty acid cycling"). Transport across the proton channel along a chain of hydrogen bonds (Grotthus mechanism) may include fatty acid carboxyl groups or occur in the absence of fatty acids. In this work, we demonstrate that planar bilayers reconstituted with UCP1 exhibit an increase in membrane conductivity exclusively in the presence of fatty acids. Hence, we can exclude the hypothesis considering a preexisting H⁺ channel in UCP1, which does not require fatty acid for function. The augmented conductivity is nearly completely blocked by ATP. Direct application of transmembrane voltage and precise current measurements allowed determination of ATP-sensitive conductances at 0 and 150 mV as 11.5 and 54.3 pS, respectively, by reconstituting nearly 3 x 10⁵ copies of UCP1. The proton conductivity measurements carried out in presence of a pH gradient (0.4 units) allowed estimation of proton turnover numbers per UCP1 molecule. The observed transport rate of 14 s^–1 is compatible both with carrier and channel nature of UCP1.

Received for publication, April 9, 2003 , and in revised form, June 6, 2003.

^* This work was supported by the Deutsche Forschungsgemeinschaft (Grants Po-524/2-1 (to E. E. P.) and Po-533/7-1 (to P. P.) and the Academy of Sciences of the Czech Republic (Grant A5011106-AVOZ5011922). 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: Neuroscience Research Center, Medical Department Charité, Humboldt University, Schumannstr. 20/21, 10117 Berlin, Germany. Tel.: 49-30-450-528141; Fax: 49-30-450-576904; E-mail: elena.pohl{at}charite.de.

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