Voltage-clamp fluorometry in the local environment of the C255-C511 disulfide bridge of the Na+/glucose cotransporter |
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Authors: | Gagnon Dominique G Frindel Carole Lapointe Jean-Yves |
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Affiliation: | Groupe d'étude des protéines membranaires (GEPROM), Université de Montréal, Montréal, Québec, Canada. |
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Abstract: | 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 (DeltaF) 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 alphaMG (a nonmetabolizable glucose analog) concentrations. A progressive increase in alphaMG concentration drastically changed the maximal voltage-dependent DeltaF and produced a positive shift in the midpoint of the DeltaF-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 alphaMG substrate significantly modifies the environment of C255 and C511. |
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Keywords: | Alexa488, Alexa Fluor 488 C5 maleimide αMG:, α-methyl-glucose TMR5M-C255, C255 labeled by TMR5M FM, fluorescein-5-maleimide ΔF-Vm, fluorescence change versus membrane potential hSGLT1, human isoform of SGLT1 I-V, current versus membrane potential Icotr, αMG-induced cotransport current (IαMG-I90Na) mmlsi1" onclick=" submitCitation('/science?_ob=MathURL& _method=retrieve& _eid=1-s2.0-S0006349507710457& _mathId=si1.gif& _pii=S0006349507710457& _issn=00063495& _acct=C000054348& _version=1& _userid=3837164& md5=c098a38d06d2144c5c052348e7b9b2c5')" style=" cursor:pointer " alt=" Click to view the MathML source" title=" Click to view the MathML source" > 15" border=" 0" style=" vertical-align:bottom" width=" 37" alt=" View the MathML source" title=" View the MathML source" src=" http://ars.els-cdn.com/content/image/1-s2.0-S0006349507710457-si1.gif" >, apparent affinity for αMG Pz, phlorizin Q-V, transferred charge versus membrane potential SGLT1, high affinity Na+/glucose cotransporter TMS, transmembrane segment TMR5M or TMR6M, tetramethylrhodamine-5(or 6)-maleimide VCF, voltage-clamp fluorometry Vm, membrane potential wt, wild-type |
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