Abstract
The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca(2+), Mg(2+), and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca(2+) stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca(2+), and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg(2+) present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836-839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion.
Highlights
The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane
(i) the single channels are exquisitely sensitive to inhibition by intracellular Ca2؉, and (ii) both the store-operated current and the singlechannel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane
During whole cell measurements in Jurkat T-lymphocytes, recently, Kerschbaum and Cahalan [11] were able to detect for the first time single-channel events which they attributed to CRAC channels
Summary
The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca2؉ stores, whether in excised patches or in whole cell mode. During whole cell measurements in Jurkat T-lymphocytes, recently, Kerschbaum and Cahalan [11] were able to detect for the first time single-channel events which they attributed to CRAC channels. Their approach was to measure currents in the complete absence of intra- and extracellular divalent cations. We report similar single-channel activities measured for the first time in excised plasma membrane patches, using a mast cell line (RBL-2H3 m1) and divalent cation-free solutions. This report offers new insight into the possible regulation mechanism of native SOCs and provides a novel approach to investigate these channels under low divalent conditions
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