Regulation of CRAC Channel Activity by Recruitment of Silent Channels to a High Open-probability Gating Mode

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 K_i 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 (P_o) of ∼0.8. By extrapolating the variance/mean current ratio to the condition of full blockade (P_o = 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 I_CRAC 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.