On the characterisation of the mechanism underlying passive activation of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia cells.
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 (ICRAC) 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 ICRAC, 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 ICRAC 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 > or = 16,670 were necessary to fully activate ICRAC. 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 ICRAC from activating passively, and are responsible for the phasic nature of the current, its time course of development and its overall extent.
Fierro
L
L
Laboratory of Molecular and Cellular Signalling, Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK.
Parekh
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AB
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015471:Leukemia, Basophilic, Acute; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D051381:Rats; D012430:Ruthenium Red; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002614:Chelating Agents; D007211:Indoles; D012430:Ruthenium Red; D004533:Egtazic Acid; D006493:Heparin; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; C000543:cyclopiazonic acid