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Vol. 19, Issue 10, 4341-4351, October 2008

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Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells

Monica Carmosino^*, Ignacio Giménez^*,, Michael Caplan^*,, and Biff Forbush^*,

*Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520

Submitted May 14, 2008; Revised July 16, 2008; Accepted July 17, 2008
Monitoring Editor: Keith E. Mostov

The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the "secretory" NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.

These authors contributed equally to this work.

Present address: Instituto Aragones de Ciencias de la Salud, 50009 Zaragoza, Spain.

Address correspondence to: Monica Carmosino (monica.carmosino{at}yale.edu)

Abbreviations used: HA, hemagglutinin; HEK, human embryonic kidney; MDCK, Madin-Darby canine kidney; NKCC, Na-K-Cl cotransporter; PLAP, placental alkaline phosphatase; TM, transmembrane domain; VSVG, vesicular stomatitis virus glycoprotein.