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

摘要

class="enumerated" style="list-style-type:decimal">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 (ICRAC) in rat basophilic leukaemia (RBL-1) cells under conditions of weak cytoplasmic Ca²⁺ buffering.Dialysis with a maximal concentration of inositol 1,4,5-trisphosphate (IP3) routinely failed to activate macroscopic ICRAC 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 ICRAC in low Ca²⁺ buffering conditions.Dialysis with the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump blocker thapsigargin was able to activate ICRAC 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 ICRAC in low buffer.Following activation of ICRAC by intracellular dialysis with IP3 and thapsigargin in low buffer, the current was very selective for Ca²⁺ (apparent KD of 1 mM). Sr²⁺ and Ba²⁺ were less effective charge carriers and Na⁺ was not conducted to any appreciable extent. The ionic selectivity of ICRAC was very similar in low or high intracellular Ca²⁺ buffer.Fast Ca²⁺-dependent inactivation of ICRAC 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 ICRAC cannot be seen under conditions of low cytoplasmic Ca²⁺ buffering.ICRAC 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.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.