CRAC (calcium release-activated Ca2+) channels attain an extremely high selectivity for Ca2+ from the blockade of monovalent cation permeation by Ca2+ within the pore. In this study we have exploited the blockade by Ca2+ to examine the size of the CRAC channel pore, its unitary conductance for monovalent cations, and channel gating properties. The permeation of a series of methylammonium compounds under divalent cation-free conditions indicates a minimum pore diameter of 3.9 Å. Extracellular Ca2+ blocks monovalent flux in a manner consistent with a single intrapore site having an effective Ki of 20 μM at -110 mV. Block increases with hyperpolarization, but declines below -100 mV, most likely due to permeation of Ca2+. Analysis of monovalent current noise induced by increasing levels of block by extracellular Ca2+ indicates an open probability (P0) of ∼0.8. By extrapolating the variance/mean current ratio to the condition of full blockade (P0 = 0), we estimate a unitary conductance of ∼0.7 pS for Na+, or three to fourfold higher than previous estimates. Removal of extracellular Ca2+ causes the monovalent current to decline over tens of seconds, a process termed depotentiation. The declining current appears to result from a reduction in the number of active channels without a change in their high open probability. Similarly, low concentrations of 2-APB that enhance ICRAC increase the number of active channels while open probability remains constant. We conclude that the slow regulation of whole-cell CRAC current by store depletion, extracellular Ca2+, and 2-APB involves the stepwise recruitment of silent channels to a high open-probability gating mode.
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