Abstract
Feedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2+]i measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of approximately 30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EFTA in the recording pipette that prevented increases in [Ca2+]i. Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by approximately 50%, indicating that store refilling normally contributes significantly to this process. The store-independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, and by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation. We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor-generated Ca2+ signals.
Highlights
Feedback regulation of Ca2 + release-activated Ca2 + (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2 +] ; measurement techniques
Under conditions of moderate intracellular Ca2 + buffering, elevating [Ca2 +]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of -30 s, This slow inactivation was evoked by a rise in [Ca2 +];, as it was effectively suppressed by an elevated level ofEGTA in the recording pipette that prevented increases in [Ca2 + ]i ' Blockade of Ca2 + uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by -50%, indicating that store refilling normally contributes significantly to this process
Fast and Slow Inactivation of ICRAC by Intracellular Ca2 + I CRAC was induced in Jurkat leukemic T cells by the passive depletion of intracellular Ca2+ stores
Summary
Feedback regulation of Ca2 + release-activated Ca2 + (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2 +] ; measurement techniques. Is the primary mechanism by which cell surface receptors activate Ca 2 + influx across the plasma membrane [1] This phenomenon was first proposed by Putney and termed capacitative Ca 2+ entry [2]. Randriamampita and Tsien [12] isolated a fraction from the cytoplasm of stimulated Jurkat leukemic human T cells that triggers Ca2 + influx when applied to the exterior of astrocyte, neuroblastoma, and macrophage cell lines, apparently without releasing Ca2 + from stores This activity was attributed to calcium influx factor, a small «500 Mr ) nonproteinaceous phosphate-containing factor that they proposed as a diffusible messenger responsible for activating capacitative Ca2 + entry. The presence of additional negative feedback pathways through which Ca 2 + may turn off CRAC channels has not been explored
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