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Related Experiment Videos

Conformational changes couple Na+ and glucose transport

D D Loo1, B A Hirayama, E M Gallardo

  • 1Department of Physiology, University of California Los Angeles School of Medicine, Center for the Health Sciences, Los Angeles, CA 90095-1751, USA. dloo@physiology.medsch.ucla.edu

Proceedings of the National Academy of Sciences of the United States of America
|June 24, 1998
PubMed
Summary

Cotransport proteins use ligand-induced conformational changes to move substrates across membranes. Studies on the Na+/glucose cotransporter (SGLT1) reveal specific structural shifts critical for sugar translocation, linking protein movement to ion transport.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Membrane Transport

Background:

  • The precise mechanism of cotransport proteins, which couple the movement of multiple substrates across cell membranes, remains largely unknown.
  • A prevailing hypothesis suggests ligand-induced conformational changes alter substrate-binding site accessibility between membrane faces.

Purpose of the Study:

  • To investigate the proposed model of ligand-induced conformational changes in cotransport.
  • To elucidate the role of specific residues in the Na+/glucose cotransporter (SGLT1) during substrate translocation.

Main Methods:

  • Site-directed mutagenesis to introduce a cysteine residue (Q457C) in the putative sugar-translocation domain of SGLT1.
  • Covalent labeling of the Q457C residue with methanethiosulfonate reagents to assess accessibility.

Related Experiment Videos

  • Measurement of sugar transport, binding, and presteady-state charge movement using electrophysiological techniques and fluorescence.
  • Voltage-jump experiments on rhodamine-6-maleimide-labeled Q457C mutants.

    • Main Results:

    • The SGLT1 Q457C mutant retained sugar transport activity, which was abolished by alkylation.
    • Alkylation specifically inhibited sugar translocation, not sugar binding, indicating Q457's role in movement.
    • Accessibility of Q457C to alkylating agents was dependent on external Na+, and inhibited by external sugar and phlorizin.
    • The accessibility of Q457C exhibited voltage dependence directly correlated with SGLT1's presteady-state charge movement.

    Conclusions:

    • Conformational changes are indeed responsible for coupling Na+ and sugar transport in SGLT1.
    • The residue at position 457 (Q457) plays a crucial role in the translocation of sugar across the membrane.
    • These findings support the model of ligand-induced conformational transitions driving cotransporter function.