Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 1051K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Loo, D. D.F. Articles by Wright, E. M. Search for Related Content PubMed PubMed Citation Articles by Loo, D. D.F. Articles by Wright, E. M. Social Bookmarking                      What's this?

Department of Physiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA 90095

Correspondence to Donald Loo: dloo{at}mednet.ucla.edu

This study examines the conformations of the Na⁺/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na⁺ buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants _med (3–20 ms, medium) and _slow (15–70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (F). The charge vs. voltage (Q-V) and fluorescence vs. voltage (F-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: z (apparent valence of voltage sensor) 1; and V_0.5 (midpoint voltage) between –15 and –40 mV. Sugar induced an inward current (Na⁺/glucose cotransport), and reduced maximal charge (Q_max) and fluorescence (F_max) with half-maximal concentrations (K_0.5) of 1 mM. Increasing [MDG]_o also shifted the V_0.5 for Q and F to more positive values, with K_0.5's 1 mM. The major difference between Q and F was that at saturating [MDG]_o, the presteady-state current (and Q_max) was totally abolished, whereas F_max was only reduced 50%. Phlorizin reduced both Q_max and F_max (K_i 0.4 µM), with no changes in V_0.5's or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na⁺/sugar cotransport (saturating voltage and external Na⁺ and sugar concentrations) is the slow release of Na⁺ from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.

A.-K. Meinild's present address is August Krogh Institute, University of Copenhagen, Universitetsparken 13, Copenhagen 2100, Denmark.

Abbreviations used in this paper: au, arbitrary unit of fluorescence intensity; SGLT1, Na⁺/glucose cotransporter; hSGLT1, human Na⁺/glucose cotransporter; MDG, -methyl-D-glucopyranoside; TMR6M, tetramethylrhodamine-6-maleimide; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Weyand, T. Shimamura, S. Yajima, S. Suzuki, O. Mirza, K. Krusong, E. P. Carpenter, N. G. Rutherford, J. M. Hadden, J. O'Reilly, et al. Structure and Molecular Mechanism of a Nucleobase-Cation-Symport-1 Family Transporter Science, October 31, 2008; 322(5902): 709 - 713. [Abstract] [Full Text] [PDF]

				S. Faham, A. Watanabe, G. M. Besserer, D. Cascio, A. Specht, B. A. Hirayama, E. M. Wright, and J. Abramson The Crystal Structure of a Sodium Galactose Transporter Reveals Mechanistic Insights into Na+/Sugar Symport Science, August 8, 2008; 321(5890): 810 - 814. [Abstract] [Full Text] [PDF]

				K. Erreger, C. Grewer, J. A. Javitch, and A. Galli Currents in Response to Rapid Concentration Jumps of Amphetamine Uncover Novel Aspects of Human Dopamine Transporter Function J. Neurosci., January 23, 2008; 28(4): 976 - 989. [Abstract] [Full Text] [PDF]

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents