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The Na+-phosphate cotransport system (NaPi-II) with a cleaved protein backbone: implications on function and membrane

B Kohl1, C A Wagner, B Huelseweh

  • 1Max-Planck-Institut fur molekulare Physiologie, Abteilung Epithelphysiologie, Rheinlanddamm 201, 44139 Dortmund, Germany.

The Journal of Physiology
|May 22, 1998
PubMed
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The NaPi-II inorganic phosphate transporter requires specific fragments (1-3 and 4-8) to function. These fragments, when combined, restore phosphate transport and localize to the oocyte membrane, indicating their essential role in transporter activity.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Physiology

Background:

  • Renal inorganic phosphate (Pi) reabsorption relies on the Na+-Pi cotransporter (NaPi-II).
  • NaPi-II is thought to possess eight transmembrane domains and undergo in vivo proteolytic cleavage.
  • Understanding NaPi-II structure-function is crucial for renal physiology.

Purpose of the Study:

  • To investigate the functional consequences of interrupting the NaPi-II transporter backbone.
  • To identify essential domains for NaPi-II cotransporter activity and localization.

Main Methods:

  • Construction of complementary N- and C-terminal cDNA fragments of winter flounder NaPi-II.
  • Expression of NaPi-II fragments in Xenopus oocytes.
  • Measurement of inorganic phosphate flux.

Related Experiment Videos

  • Immunohistochemistry on oocyte cryosections and intact oocytes.

    • Main Results:

    • Individual NaPi-II fragments or the 1-5 plus 6-8 combination did not restore Pi flux.
    • Coexpression of fragments 1-3 plus 4-8 restored NaPi-II transport activity, with similar pH dependency and binding kinetics but lower maximal rate (vmax).
    • Immunohistochemistry confirmed the membrane localization of the functional 1-3 plus 4-8 fragment combination.

    Conclusions:

    • The NaPi-II transporter's function depends on the integrity of specific domains, particularly the combination of fragments 1-3 and 4-8.
    • The loop between transmembrane domains 3 and 4 contains extracellular epitopes crucial for transporter function and localization.