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

Leonardo Fierro and Anant B. Parekh

1. Tight-seal whole-cell patch clamp experiments were performed to investigate the mechanism whereby passive depletion of stores activates the Ca²⁺ release-activated Ca²⁺ current (I_CRAC) in rat basophilic leukaemia (RBL) cells.

2. Passive depletion of stores was achieved by dialysing cells with different concentrations of Ca²⁺ chelators. Low concentrations generally evoked a submaximal I_CRAC, which developed slowly and monophasically. Higher concentrations 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 16 670 were necessary to fully activate I_CRAC. Because the Ca²⁺ binding ratio within the stores is presumably low, this indicates that other factors like Ca²⁺ transport across the stores membrane are rate limiting for passive store depletion.

5. Heparin and Ruthenium Red both failed to affect passive Ca²⁺ leak from the intracellular stores.

6. Treatment with sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump blockers 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 of the current, its time course of development and its overall extent.

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