A calmodulin activated Ca 2+-dependent K + channel in human erythrocyte membrane inside-out vesicles

Abstract

The role of calmodulin in stimulating active calcium transport in the human red cell membrane is well documented. In contrast, efforts to characterize the effect of calmodulin on the Ca 2+-dependent K + channel in erythrocyte membranes have given rise to conflicting reports. These studies have indicated that experimental conditions may play a critical role in preserving the Ca 2+-dependent K + channels in erythrocyte inside-out vesicles. With these observations in mind, a double-labelling study of simultaneous active Ca 2+ and passive Rb + uptake in red-cell inside-out vesicles was undertaken. Addition of calmodulin and ATP to a suspension of inside-out vesicles containing 1 mM K + caused a Ca 2+-dependent increase in both the rate of active calcium transport and Rb + uptake. The initial Rb + isotope flux was increased 3-fold over the rate observed in the absence of calmodulin. The k 1 2 for activation of K + permeability was approx. 5 · 10 −7 M Ca 2+ as compared to 10 −6 M Ca 2+ for active Ca 2+ transport. Addition of the calmodulin antagonists pimozide and chlorpromazine blocked calmodulin activation of the Ca 2+-dependent K + channel. The observation that activation of the K + channel occurs at Ca 2+ concentrations which are lower than those required for maximum stimulation of the calcium pump suggests that these processes are dependent on two states of the calmodulin molecule, characterized by a lower or higher amount of Ca 2+ bound to calmodulin.