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Functional studies on a split type II Na/P(i)-cotransporter.

C Ehnes1, I C Forster, K Köhler

  • 1Institute of Physiology, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

The Journal of Membrane Biology
|August 16, 2002
PubMed
Summary
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The type IIa Na/P(i)-cotransporter can be cleaved in its extracellular loop and still function. This split transporter, composed of p40 and p45 domains, remains active and properly localized in the plasma membrane.

Area of Science:

  • Molecular biology
  • Renal physiology
  • Membrane transport

Background:

  • The type IIa Na/P(i)-cotransporter is crucial for phosphate reabsorption in the proximal tubule.
  • Evidence suggests this transporter undergoes cleavage in its large extracellular loop (ECL-2).

Purpose of the Study:

  • To investigate the functional properties and membrane distribution of the cleaved NaP(i)-IIa transporter.
  • To determine if cleavage affects the transporter's cotransport function.

Main Methods:

  • Expression of complementary transporter parts (p40 and p45) in Xenopus laevis oocytes.
  • In vitro cleavage of the wild-type (WT) protein using trypsin.
  • Analysis of protein localization and functional transport assays.

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Main Results:

  • Both expression of complementary parts and trypsin cleavage resulted in a split NaP(i)-IIa protein in the plasma membrane.
  • The two domains are likely linked by a disulfide bridge involving cysteines 306 and 334.
  • Surface expression and cotransport function were retained in the cleaved transporter, with identical characteristics to the WT protein.

Conclusions:

  • Cleavage of the type IIa Na/P(i)-cotransporter at ECL-2 is compatible with its cotransport function.
  • The split transporter remains functional and correctly localized in the plasma membrane.
  • The integrity of both domains is essential for surface expression.