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

Abstract

We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegansK-Cl 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-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share ∼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 10 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-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.

Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration

“…While staurosporine can induce apoptosis only at high concentrations, at lower concentrations it has been shown to inhibit the protein kinases PKC, PKA, PKG, CAMKII, and MLCK, as well as other kinases in a concentration-dependent manner, and to stimulate K-Cl cotransport in red blood cells (25,(35)(36)(37). Because staurosporine's strongest inhibitory effect is on PKC kinases, and in C. elegans it has been shown to inhibit PKC activity (38), we tested whether it exerts its effects on regrowing neurites via the inhibition of this particular kinase.…”

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

“…This alkaloid acts as a kinase inhibitor that activates KCCs (33,60). Staurosporine closely mimicked the effect of NEM.…”

A Dominant Negative Mutant of the KCC1 K-Cl Cotransporter

“…In addition, each mKCC1 truncation mutant examined in the current work can be covalently cross-linked to the homodimeric state. 6 Thus, the presence of neither cytoplasmic domain of mKCC1 is required for homo-oligomerization.…”

“…Even in isotonic conditions, 86 Rb ϩ influx was higher in oocytes expressing mKCC1 than in water-injected oocytes (p Ͻ 0.001). Our previous studies of mKCC1 in Xenopus oocytes (7) and other studies of rabbit (5,19) and human KCC1 in 293 cells (6) and of native Xenopus oocyte K-Cl cotransport (14) have shown that this activated 86 Rb ϩ influx requires bath chloride and is inhibited by the serine-threonine phosphatase inhibitors okadaic acid and calyculin and by the diuretic diindenylalkanoic acid. Removal of only the eight C-terminal residues from the C-terminal cytoplasmic tail of mKCC1, as in ⌬ c 1077, preserved a low level of activity in isotonic medium (p ϭ 0.008) but abolished stimulation of 86 Rb uptake elicited by hypotonicity.…”

“…K-Cl cotransporters also serve to regulate the cellular electrochemical equilibrium potential for Cl Ϫ and can regulate [K ϩ ] of the interstitial space, especially in the nervous system. KCC1 cDNAs have been cloned from rabbits (5), rats (5), humans (5), pigs (6), and mice (7,8), with gene structures reported for humans (6), mice (9), and Caenorhabditis elegans (6). KCC2 cDNA has been cloned from rat (10).…”