Characterisation of three pathways for osmolyte efflux in human erythroleukemia cells

Cell volume decrease is a key step during differentiation of erythroid cells. This could arise from membrane transporter activation leading to a loss of cell osmolytes; however, the pathways involved are poorly understood. We have characterised Cl−-independent K+ and 3H-taurine efflux from the erythroleukemia cell line, K562. K+ efflux (measured using 86Rb+) from pre-loaded cells subjected to hypo-osmotic challenge demonstrated two phases, a rapid increase in K+ efflux followed by a smaller slower increase. Swelling-activated taurine efflux only demonstrated a single phase. Both phases of K+ efflux were significantly (P < 0.05) blocked by anion channel inhibitor 5-nitro-2-(3-phenypropylamino)-benzoic acid (NPPB). However the antiestrogen, tamoxifen, only inhibited the slow late phase. The initial rapid phase had a higher IC50 for NPPB inhibition than the slow phase, and was insensitive to protein kinases inhibitors KN-62, wortmannin and PD98059. For the slow K+ efflux phase, the IC50 for NPPB inhibition and the inhibition by KN-62, wortmannin, genistein or PD98059, were very similar to those measured for the hypo-osmotically-activated taurine efflux. With NPPB (100 μM) present, the slow K+ efflux phase was further significantly decreased by the Ca2+ chelator BAPTA-AM or by the Ca2+-activated K+ channel blockers clotrimazole and charybdotoxin but not by apamin. Thus, at least 3 Cl−-independent pathways are involved: (a) a tamoxifen-sensitive and taurine-permeable anion channel; (b) a tamoxifen-insensitive and taurine-impermeable K+ efflux pathway; and (c) a subtype of Ca2+-activated K+ channel. Any or all of these could be involved in the cell volume decrease associated with differentiation in K562 cells.