Substantial depletion of the intracellular Ca2+ stores is required for macroscopic activation of the Ca2+ release‐activated Ca2+ current in rat basophilic leukaemia cells

Abstract

1 Tight-seal whole-cell patch clamp experiments were performed to examine the ability of different intracellular Ca²⁺ mobilising agents to activate the Ca²⁺ release-activated Ca²⁺ current (I_CRAC) in rat basophilic leukaemia (RBL-1) cells under conditions of weak cytoplasmic Ca²⁺ buffering. 2 Dialysis with a maximal concentration of inositol 1,4,5-trisphosphate (IP₃) routinely failed to activate macroscopic I_CRAC in low buffer (0.1 mM EGTA, BAPTA or dimethyl BAPTA), whereas it activated the current to its maximal extent in high buffer (10 mM EGTA). Dialysis with a poorly metabolisable analogue of IP₃, with ionomycin, or with IP₃ and ionomycin all failed to generate macroscopic I_CRAC in low Ca²⁺ buffering conditions. 3 Dialysis with the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump blocker thapsigargin was able to activate I_CRAC even in the presence of low cytoplasmic Ca²⁺ buffering, albeit at a slow rate. Exposure to IP₃ together with the SERCA blockers thapsigargin, thapsigargicin or cyclopiazonic acid rapidly activated I_CRAC in low buffer. 4 Following activation of I_CRAC by intracellular dialysis with IP₃ and thapsigargin in low buffer, the current was very selective for Ca²⁺ (apparent K_D of 1 mM). Sr²⁺ and Ba²⁺ were less effective charge carriers and Na⁺ was not conducted to any appreciable extent. The ionic selectivity of I_CRAC was very similar in low or high intracellular Ca²⁺ buffer. 5 Fast Ca²⁺-dependent inactivation of I_CRAC occurred at a similar rate and to a similar extent in low or high Ca²⁺ buffer. Ca²⁺-dependent inactivation is not the reason why macroscopic I_CRAC cannot be seen under conditions of low cytoplasmic Ca²⁺ buffering. 6 I _CRAC could be activated by combining IP₃ with thapsigargin, even in the presence of 100 μM Ca²⁺ and the absence of any exogenous Ca²⁺ chelator, where ATP and glutamate represented the only Ca²⁺ buffers in the pipette solution. 7 Our results suggest that a threshold exists within the IP₃-sensitive Ca²⁺ store, below which intraluminal Ca²⁺ needs to fall before I_CRAC activates. Possible models to explain the results are discussed.