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Originally published as Biophys J. BioFAST on January 5, 2007.
doi:10.1529/biophysj.106.097964
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Biophysical Journal 92:2403-2411 (2007)
© 2007 The Biophysical Society

Voltage-Clamp Fluorometry in the Local Environment of the C255–C511 Disulfide Bridge of the Na+/Glucose Cotransporter

Dominique G. Gagnon, Carole Frindel and Jean-Yves Lapointe

Groupe d'étude des protéines membranaires (GÉPROM), Université de Montréal, Montréal, Québec, Canada

Correspondence: Address reprint requests to Jean-Yves Lapointe, Groupe d'étude des protéines membranaires (GÉPROM), Université de Montréal, C.P. 6128, succ. Centre-ville, Montréal, Québec H3C 3J7, Canada. Tel.: 514-343-7046; Fax: 514-343-7146; E-mail: jean-yves.lapointe{at}umontreal.ca.

We recently identified a functionally important disulfide bridge between C255 and C511 of the human Na+/glucose cotransporter SGLT1. In this study, voltage-clamp fluorometry was used to characterize the fluorescence of four different dyes attached to C255 and C511 under various ionic and substrate/inhibitor conditions. State-dependent fluorescence changes ({Delta}F) were observed when TMR5M or TMR6M dyes were attached to C255 and C511 or when Alexa488 was bound to C511. TMR5M-C511 was extremely sensitive to membrane potential (Vm) and to external Na+ and {alpha}MG (a nonmetabolizable glucose analog) concentrations. A progressive increase in {alpha}MG concentration drastically changed the maximal voltage-dependent {Delta}F and produced a positive shift in the midpoint of the {Delta}F-Vm curve. By determining specific fluorescence intensity for each state of the cotransporter, our steady-state fluorescence data could be reproduced using the rate constants previously proposed for a five-state kinetic model exclusively derived from electrophysiological measurements. Our results bring an independent support to the proposed kinetic model and show that the binding of {alpha}MG substrate significantly modifies the environment of C255 and C511.






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Copyright © 2007 by the Biophysical Society.