Topology of the type IIa Na+/P(i) cotransporter

J Membr Biol. 2006;212(1):41-9. doi: 10.1007/s00232-006-0033-2. Epub 2007 Jan 6.

Abstract

The type IIa Na(+)/P(i) cotransporter (NaPi-IIa) plays a key role in the reabsorption of inorganic phosphate (P(i)) in the renal proximal tubule. The rat NaPi-IIa isoform is a protein of 637 residues for which different algorithms predict 8-12 transmembrane domains (TMDs). Epitope tagging experiments demonstrated that both the N and the C termini of NaPi-IIa are located intracellularly. Site-directed mutagenesis revealed two N-glycosylation sites in a large putative extracellular loop. Results from structure-function studies suggested the assembly of two similar opposed regions that possibly constitute part of the substrate translocation pathway for one phosphate ion together with three sodium ions. Apart from these topological aspects, other structural features of NaPi-IIa are not known. In this study, we have addressed the topology of NaPi-IIa using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NaPi-IIa TMDs. Based on the results of in vitro transcription/translation analyses, we propose a model of NaPi-IIa comprising 12 TMDs, with both N and C termini orientated intracellularly and a large hydrophilic extracellular loop between the fifth and sixth TMDs. The proposed model is in good agreement with the prediction of the NaPi-IIa structure obtained by the hidden Markov algorithm HMMTOP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Extracellular Space / chemistry
  • Extracellular Space / genetics
  • Extracellular Space / metabolism
  • Glycosylation
  • Intracellular Fluid / chemistry
  • Intracellular Fluid / metabolism
  • Models, Molecular
  • Oocytes / metabolism
  • Phosphate Transport Proteins / biosynthesis
  • Phosphate Transport Proteins / chemistry*
  • Phosphate Transport Proteins / genetics
  • Rats
  • Sodium-Phosphate Cotransporter Proteins, Type IIa / biosynthesis
  • Sodium-Phosphate Cotransporter Proteins, Type IIa / chemistry*
  • Sodium-Phosphate Cotransporter Proteins, Type IIa / genetics
  • Transcription, Genetic
  • Transfection
  • Xenopus laevis / genetics

Substances

  • Phosphate Transport Proteins
  • Sodium-Phosphate Cotransporter Proteins, Type IIa