On the characterisation of the mechanism underlying passive activation of the Ca2+ release-activated Ca2+ current I-CRAC in rat basophilic leukaemia cells

1. Tight-seal whole-cell patch clamp experiments were performed to investig ate the mechanism whereby passive depletion of stores activates the Ca2+ re lease-activated Ca2+ current I-CRAC in rat basophilic leukaemia (RBL) cells . 2. Passive depletion of stores was achieved Ly dialysing cells with differe nt concentrations of Ca2+ chelators. Low concentrations generally evoked a submaximal I-CRAC which developed slowly and monophasically. Higher concent rations resulted in a biphasic current in which the initial slow monophasic component developed into a faster and bigger second phase. 3. The kinetics of I-CRAC as well as its final amplitude were not affected by Ca2+ chelators that had different affinities or speeds of binding. 4.Exogenous Ca2+ binding ratios greater than or equal to 16 670 were necess ary to fully activate I-CRAC. Because the Ca2+ binding ratio within the sto res is presumably lon, this indicates that other factors like Ca2+ transpor t across the stores membrane are rate limiting for passive store depletion. 5. Heparin and Ruthenium Red both failed to affect passive Ca2+ leak from t he intracellular stores. 6. Treatment with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump bloc kers dramatically altered the kinetics of activation of biphasic currents, and increased the amplitude of monophasic ones. 7. Our results suggest that SERCA pumps are very effective in preventing I- CRAC from activating passively, and are responsible for the phasic nature o f the current, its time course of development and its overall extent.