title: Functional expression of human NKCC1 from a synthetic cassette-based cDNA: introduction of extracellular epitope tags and removal of cysteines.
abstract: The Na-K-Cl cotransporter (NKCC) couples the movement of Na(+), K(+), and Cl(-) ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(-) -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.
authors: Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut, United States of America.
authors: Suma
authors: S
authors: Somasekharan
authors: Michelle Y
authors: MY
authors: Monette
authors: Biff
authors: B
authors: Forbush
mesh_terms: D003545:Cysteine; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000096:biosynthesis* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D064506:Solute Carrier Family 12, Member 2 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D000939:Epitopes; D011993:Recombinant Fusion Proteins; C578877:SLC12A2 protein, human; D064506:Solute Carrier Family 12, Member 2; D003545:Cysteine