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J. Biol. Chem., Vol. 281, Issue 39, 28837-28849, September 29, 2006

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Mapping Conformational Changes of a Type IIb Na⁺/P_i Cotransporter by Voltage Clamp Fluorometry^*

From the Institute for Physiology and the Center for Integrative Human Physiology (ZIHP), University of Zurich, CH-8057 Zurich, Switzerland

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na⁺/P_i cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na⁺, Li⁺, and P_i-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na⁺ and Li⁺. Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.

Received for publication, April 21, 2006 , and in revised form, July 5, 2006.

^* This work was supported in part by the Gebert Rüf Foundation and the Swiss National Science Foundation (to H. M.). 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.

¹ To whom correspondence may be addressed: Institute of Physiology, University of Zurich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Tel.: 41-44-635-5059; Fax: 41-44-635-5615; E-mail: leilav{at}physiol.unizh.ch. ² To whom correspondence may be addressed. E-mail: IForster{at}access.unizh.ch.

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