Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endog- enous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and map ped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised ag ainst recombinant mKCC1 function as immunoblot and immunoprecipitation reag ents. The tissue distributions of mKCC1 mRNA and protein are widespread, an d mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes o f multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticuloc yte counts in density-fractionated cells, in bleeding-induced reticulocytos is, in mouse models of sickle cell disease and thalassemia, and in the corr esponding human disorders. mKCC1-mediated uptake of Rb-86 into Xenopus oocy tes requires extracellular Cl-, is blocked by the diuretic R(+)-[2-n-butyl- 6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy] acetic a cid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite st udies of KCC1 structure-function relationships and of the pathobiology of K CC1-mediated K-Cl cotransport.