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Relaxation kinetics of the Na+/glucose cotransporter.

Authors
  • Loo, D D
  • Hazama, A
  • Supplisson, S
  • Turk, E
  • Wright, E M
Type
Published Article
Journal
Proceedings of the National Academy of Sciences
Publisher
Proceedings of the National Academy of Sciences
Publication Date
Jun 15, 1993
Volume
90
Issue
12
Pages
5767–5771
Identifiers
PMID: 8516326
Source
Medline
License
Unknown

Abstract

An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Qmax of approximately 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Qmax. Lowering external Na+ from 100 to 10 mM reduced Qmax 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Qmax was independent of, but tau was dependent on, temperature (a 10 degrees C increase increased tau by a factor of approximately 2.5 at -50 mV). Addition of sugar or phlorizin reduced Qmax. Analyses of hSGLT1 pre-steady-state kinetics indicate that transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Qmax provides a measure of transporter density (approximately 10(4)/microns 2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechanisms.

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