Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

摘要: Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. regulation poorly understood because its restricted distribution difficulties with expression mammalian cultures. Here we compare phosphorylation N-termini cotransporters, measured phospho-specific antibodies, bumetanide-sensitive transport K+ (86Rb+) (activity) HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities transfected transporters distinguished those endogenous ones by working at 37°C. activities highest after pre-incubation hypotonic low-[Cl−] media to reduce [Cl−] volume during flux measurement. Phosphorylation both more than doubled. Pre-incubation ouabain also strongly stimulated substantially increased phosphorylation, whereas Na+-free maximally doubled but inhibited fNKCC2A, small increase phosphorylation. Kinase inhibitors halved activity inhibition phosphatases calyculin A slightly fNCCC2A. Thus kinase reduced transport, stimulation was seen when increased, did not always This suggests determines transporters' potential capacity move ions, final depends on other factors. Transport cannot be reliably inferred solely using antibodies whole-cell lysates.