Potassium Influx Pathway Regulation and KCC Isoforms in Human Lens Epithelial Cells and Tissue

Abstract

Abstract: : Purpose: Identify K influx pathways [Na/K pump, Na–K–2Cl cotransport (NKCC) and K–Cl cotransport (KCC)], their regulation and KCC isoforms in human lens epithelial cells. Methods: Major K influx pathways and their response to N–ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors characterized in human lens epithelial B3 (HLEB3) cells with Rb as K congener. Reverse transcriptase polymerase chain reaction (RTPCR), Western blots and immunofluorescence identify KCC isoforms in HLEB3 cells and human lens tissue from cataract patients. Results: Ouabain (0.1 mM) and bumetanide (0.5 µM) discriminated between the Na/K pump (∼35% of total Rb influx) and NKCC (∼50%). Cl–replacement with nitrate or sulfamate detected residual [ouabain+bumetanide]–insensitive KCC (∼15%). At 0.3–0.5 mM, NEM stimulated Na/K pump two–fold (independent of external Na) and KCC up to four–fold and inhibited NKCC by ∼90%. Calyculin–A, a serine/threonine (S/T) protein phosphatase 1 inhibitor, slightly stimulated NKCC and inhibited KCC, whereas the S/T kinase inhibitor staurosporine abolished NKCC and stimulated KCC (the latter only when followed by NEM treatment). The tyrosine (Y)–kinase inhibitor genistein activated Na/K pump at >0.1 mM and abolished NKCC but did not affect KCC, whereas PP2, a tyrosine (Y)–phosphatase inhibitor, stimulated NKCC. RT–PCR revealed predicted cDNA fragments (base pairs) of KCC1 (233), KCC3a (250), KCC3b (409) and KCC4 (556) and absence of KCC2 in both HLEB3 cells and human lens tissue. Western blots with KCC1– and KCC3–specific antibodies showed expression of both isoforms in HLEB3 cells with molecular weights in predicted ranges. These antibodies and Cy3–labeled anti–IgG showed membrane immunofluorescence of both KCC1 and KCC3 contrasted with cytoplasmic staining of ß–crystallin with rabbit anti–bovine ß–crystallin antibody.Conclusions:Data suggest inverse functional regulation of NKCC and KCC by signaling cascades involving S/T– and Y– phosphorylation/dephosphorylation equilibria. Evidence for at least 3 KCC isoforms in both cultured HLEB3 cells and human lens tissue is presented. High susceptibility of thiols within these membrane transport mechanisms may play a role in UV–light–elicited photo–oxidation with consequences such as apoptosis and cataract formation

Keywords: ion transporters • signal transduction • apoptosis/cell death