RESUMO
The effects of cAMP, ATP and GTP on the Ca2(+)-dependent K+ channel of fresh (1-2 days) or cold-stored (28-36 days) human red cells were studied using atomic absorption flame photometry of Ca2(+)-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solution. When high-K+ ghosts were incubated in an isotonic Na+ medium, the rate constant of Ca2(+)-dependent K+ efflux was reduced by a half on increasing the theophylline concentration to 40 mM. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg2+ was added together with ATP, and it was abolished by raising free Ca2+ to the micromolar level. Like theophylline, isobutyl methylxanthine (10 mM) also affected K+ efflux. cAMP (0.2-0.5 mM), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K+ loss when the ghost free-Ca2+ level was below 1 microM, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2 mM) plus either theophylline (10 mM), or isobutyl methylxanthine (0.5 mM), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg2+ and it was not overcome by raising internal Ca2+. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K+ loss. Although this effect was observed in the absence of added Mg2+ (0.5 mM EDTA present), it was potentiated upon adding 2 mM Mg2+. The K+ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10 mM) or acridine orange (100 microM), while it was increased two- to fourfold by incubating with MgF2 (10 mM), or MgF2 (10 mM) + theophylline (40 mM), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5 mM GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (gamma-32P)ATP under various conditions affecting K+ channel activity, was in direct correspondence to their effect on K+ efflux. The results suggest that the K+ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.
