Abstract
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Journal of General Physiology |
| Vol/bind | 128 |
| Udgave nummer | 6 |
| Sider (fra-til) | 701-20 |
| ISSN | 0022-1295 |
| DOI | |
| Status | Udgivet - 2006 |
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I: Journal of General Physiology, Bind 128, Nr. 6, 2006, s. 701-20.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
}
TY - JOUR
T1 - Conformational Dynamics of hSGLT1 during Na+/Glucose Cotransport
AU - Loo, D. D.
AU - Hirayama, B. A.
AU - Karakossian, M. H.
AU - Meinild, A. K.
AU - Wright, E. M.
PY - 2006
Y1 - 2006
N2 - 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 V0.5 (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na+/glucose cotransport), and reduced maximal charge (Qmax) and fluorescence (Fmax) with half-maximal concentrations (K0.5) of 1 mM. Increasing [MDG]o also shifted the V0.5 for Q and F to more positive values, with K0.5's 1 mM. The major difference between Q and F was that at saturating [MDG]o, the presteady-state current (and Qmax) was totally abolished, whereas Fmax was only reduced 50%. Phlorizin reduced both Qmax and Fmax (Ki 0.4 µM), with no changes in V0.5'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.
AB - 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 V0.5 (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na+/glucose cotransport), and reduced maximal charge (Qmax) and fluorescence (Fmax) with half-maximal concentrations (K0.5) of 1 mM. Increasing [MDG]o also shifted the V0.5 for Q and F to more positive values, with K0.5's 1 mM. The major difference between Q and F was that at saturating [MDG]o, the presteady-state current (and Qmax) was totally abolished, whereas Fmax was only reduced 50%. Phlorizin reduced both Qmax and Fmax (Ki 0.4 µM), with no changes in V0.5'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.
U2 - 10.1085/jgp.200609643
DO - 10.1085/jgp.200609643
M3 - Journal article
C2 - 17130520
SN - 0022-1295
VL - 128
SP - 701
EP - 720
JO - Journal of General Physiology
JF - Journal of General Physiology
IS - 6
ER -