Regulation of potassium transport in human lens epithelial cells

The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed <10% residual [ouabain+bumetanide]-insensitive K-Cl cotransport (KCC). At 0.3-0.5 mM, NEM stimulated the Na/K pump by 2-fold independent of external Na, KCC between 2 and 4-fold, and abolished approximately 90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10 microM staurosporine, a serine/threonine kinase inhibitor, abolished NKCC, and stimulated KCC only when followed by NEM treatment. The tyrosine-kinase inhibitor genistein, at concentrations >100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.