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On the characterisation of the mechanism underlying passive activation of the Ca 2+ release‐activated Ca 2+ current I CRAC in rat basophilic leukaemia cells
Author(s) -
Fierro Leonardo,
Parekh Anant B.
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.00407.x
Subject(s) - chemistry , serca , biophysics , ruthenium red , kinetics , intracellular , membrane potential , thapsigargin , endoplasmic reticulum , patch clamp , ion transporter , atpase , calcium , biochemistry , membrane , receptor , biology , physics , organic chemistry , quantum mechanics , enzyme
1 Tight‐seal whole‐cell patch clamp experiments were performed to investigate the mechanism whereby passive depletion of stores activates the Ca 2+ release‐activated Ca 2+ current ( I CRAC ) in rat basophilic leukaemia (RBL) cells. 2 Passive depletion of stores was achieved by dialysing cells with different concentrations of Ca 2+ 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 Ca 2+ chelators that had different affinities or speeds of binding. 4 Exogenous Ca 2+ binding ratios ≥ 16 670 were necessary to fully activate I CRAC . Because the Ca 2+ binding ratio within the stores is presumably low, this indicates that other factors like Ca 2+ transport across the stores membrane are rate limiting for passive store depletion. 5 Heparin and Ruthenium Red both failed to affect passive Ca 2+ leak from the intracellular stores. 6 Treatment with sarco/endoplasmic reticulum Ca 2+ ‐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.