Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C-elegans

We isolated and characterized the cDNAs far the human, pig, and Caenorhabditis elegans K-CI cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-CI cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-CI cotransporters share similar to 45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains ID exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-CI cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-CI cotransport.