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Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane
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Exon loss accounts for differential sorting of Na-K-Cl cotransporters in polarized epithelial cells

Monica Carmosino1, Ignacio Giménez, Michael Caplan

  • 1Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA. monica.carmosino@yale.edu

Molecular Biology of the Cell
|August 1, 2008
PubMed

Insights

The renal Na-K-Cl cotransporter 2 (NKCC2) is sorted to the apical kidney membrane, while NKCC1 goes to the basolateral side. A specific exon in NKCC1

Area of Science:

  • Molecular biology
  • Cell biology
  • Physiology

Background:

  • The renal Na-K-Cl cotransporter 2 (NKCC2) is crucial for kidney function and a target for loop diuretics.
  • The related NKCC1 cotransporter is found in the basolateral membranes of various epithelia.
  • Understanding the sorting signals for these transporters is key to elucidating epithelial cell polarity.

Purpose of the Study:

  • To identify the molecular sorting signals responsible for the differential localization of NKCC1 and NKCC2.
  • To investigate the role of specific protein domains and gene structure in NKCC trafficking.

Main Methods:

  • Generation and expression of chimeric NKCC constructs in polarized renal epithelial cells.
  • Analysis of protein trafficking and localization using cell-based assays.
  • Examination of NKCC gene structure and phylogenetic analysis.

Main Results:

  • An amino acid stretch in NKCC2 was identified as an apical sorting signal.
  • A dileucine motif in the NKCC1 C terminus was found to be essential for basolateral sorting.
  • NKCC1 lacking this motif was mis-sorted to the apical membrane.
  • The dileucine motif is encoded by an exon present in NKCC1 but absent in NKCC2.

Conclusions:

  • The differential localization of NKCC1 and NKCC2 is determined by distinct sorting signals within the proteins.
  • The presence or absence of a specific exon encoding a dileucine motif is a key factor in NKCC isoform sorting.
  • Evolutionary loss of this exon in the NKCC2 gene likely accounts for its apical localization in the kidney.

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