Stable activation of single Ca2+ release-activated Ca2+ channels in divalent cation-free solutions

The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized muc osal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membra ne. The activity of the single channels in excised patches could be modulat ed by Ca2+, Mg2+, and pH. The maximal activation of these channels by dival ent cation-free conditions occurred independently of depletion of intracell ular Ca2+ stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amp lified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca2+, and (ii) bo th the store-operated current and the single-channel openings are completel y blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane, In addition, in Jurkat T cells single-channel openings with lower o pen probability have been observed in the whole cell mode with intracellula r Mg2+ present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 8 36-839), and in RBL-2H3 m1 cells a current with similar properties is activ ated by store depletion.