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

SUBMITTER: Somasekharan S 

PROVIDER: S-EPMC3855340 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature