Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies

Abstract

The Na-K-Cl cotransporter (NKCC) is present in most animal cells where it functions in cell volume homeostasis and epithelial salt transport. We developed six monoclonal antibodies (designated T4, T8, T9, T10, T12, and T14) against a fusion protein fragment encompassing the carboxy-terminal 310 amino acids of the human colonic NKCC. These T antibodies selectively recognized putative NKCC proteins in a diverse variety of animal tissues. Western blot analysis of membranes isolated from 23 types of cells identified single bands of immunoreactive protein ranging in mass from 146 to 205 kDa. The amount of immunoreactive protein detected in these cells correlated with loop diuretic binding site density. Proteins identified previously as Na-K-Cl cotransporters by loop diuretic photoaffinity labeling were mutually recognized by multiple T antibodies. Most of the T antibodies effectively immunoprecipitated the denatured form of the NKCC protein. Immunocytochemical studies on the rabbit parotid gland demonstrated that NKCC is restricted to the basolateral margin of the acinar cells and absent from the ducts, in accord with the central role of Na-K-Cl cotransport in chloride secretion. In the rabbit kidney, NKCC was localized to the apical membrane of thick ascending limb cells, consistent with its role in chloride reabsorption.

The Residues Determining Differences in Ion Affinities among the Alternative Splice Variants F, A, and B of the Mammalian Renal Na-K-Cl Cotransporter (NKCC2)

“…Oocytes (4 -6 per group) were solubilized in 100 l of homogenization buffer (1% Triton X-100), and 6 l of that homogenate were run on SDS gels, blotted, and evaluated with the T4 antibody (18).…”

Molecular components of signal amplification in olfactory sensory cilia

“…2A illustrates similar distributions of ciliary fluorescence intensities for pNKCC1 and total NKCC1, indicating that a large fraction of NKCC1 is phosphorylated. To quantify this fraction, we used Western blot analysis with the monoclonal NKCC1 antibody T4, which was shown to be reliable for this method (16). The T4 antibody stained two discrete bands at 140 kDa and 160 kDa in Western blots from olfactory epithelium membrane proteins (Fig.…”

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

“…Immunofluorescence Analysis-Visualization of sNKCC1 in transfected HEK-293 cells was performed as previously described (6). Briefly, cells were fixed in the culture dish with periodate-lysine-paraformaldehyde fixative, scraped from the dish, and pelleted at low speed; semithin cryosections were cut from cryopreserved specimens.…”

“…These proteins are all predicted to have 12 transmembrane-spanning domains that are responsible for ion transport properties (5) and large cytoplasmic N and C termini that are candidates for regulatory domains. NKCC1 is expressed in many cell types and is involved in the regulation of both cell volume and intracellular Cl Ϫ concentration (2,6). This isoform is also a major component of the basolateral membrane of secretory cells, mediating Cl Ϫ influx, the first step in the transepithelial movement of Cl Ϫ .…”