Functional studies on a split type II Na/P(i)-cotransporter.
Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/P(i)-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane distribution of this split NaP(i)-IIa transporter we followed two strategies. In one strategy we expressed the transporter as two complementary parts (p40 and p45) in Xenopus laevis oocytes and as another strategy we cleaved the WT protein with trypsin. Both strategies resulted in a split NaP(i)-IIa protein located in the plasma membrane. The two domains were tied together by a disulfide bridge, most likely involving the cysteines 306 and 334. Surface expression of the NaP(i)-IIa fragments was dependent on the presence of both domains. If both domains were coexpressed, the transporter was functional and transport characteristics were identical to those of the WT-NaP(i)-IIa protein. Corresponding to this, the transporter cleaved by trypsin also retains its transport capacity. These data indicate that cleavage of the type IIa Na/P(i)-cotransporter at ECL-2 is compatible with its cotransport function.
Ehnes
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Institute of Physiology, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
Forster
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IC
Köhler
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K
Biber
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Murer
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H
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D014357:Trypsin / Q000235:genetics / Q000494:pharmacology*; D014982:Xenopus laevis
D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; D014357:Trypsin