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Apical membrane expression of NKCC2 is directed by a domain within its cytoplasmic C‐terminus
Author(s) -
Carmosino Monica,
Gimenez Ignacio,
Caplan Michael,
Forbush Biff
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.935.4
Subject(s) - cotransporter , reabsorption , apical membrane , microbiology and biotechnology , chemistry , gene isoform , subcellular localization , n terminus , cytoplasm , kidney , biology , biochemistry , peptide sequence , membrane , gene , endocrinology , sodium , organic chemistry
The renal Na‐K‐Cl cotransporter (NKCC2) is selectively expressed in the apical membrane of thick ascending limb (TAL) cells of the mammalian kidney, where it is the central player in NaCl reabsorption, and the target of the clinically important loop diuretic drugs. In contrast, the “housekeeping” Na‐K‐Cl cotransporter (NKCC1) is localized in the basolateral membrane of many epithelia, including the distal collecting duct in the kidney. To identify the sorting signal(s) that direct apical or basolateral trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. Transient expression analysis in MDCK cells demonstrated that among the three large portions of the cotransporter (the N‐terminal, C‐terminal, and transmembrane domains) the C‐terminus of NKCC2 is necessary and sufficient for apical expression. Subsequent chimeras, in which sequential regions of the NKCC1 C‐terminus were exchanged with the corresponding regions of NKCC2 C‐terminus were stably expressed in MDCK cells. This analysis resulted in the identification of 70 aa residue stretch of sequence that is sufficient to determine the apical localization of NKCC2. The identification of sorting signals in NKCC2 will provide important tools with which to gain insight into the molecular basis of mis‐sorting diseases and the molecular basis of hypertension.