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Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305

Correspondence to Richard S. Lewis or Murali Prakriya: rslewis{at}stanford.edu; m-prakriya{at}northwestern.edu

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.

Abbreviations used in this paper: CDP, Ca²⁺-dependent potentiation; CRAC, calcium release-activated Ca²⁺; MIC, Mg²⁺-inhibited cation; Ca_V, voltage-gated Ca²⁺; TG, thapsigargin; I_CRAC, CRAC current; DVF, divalent free; P_o, open probability; 2-APB, 2-aminoethyldiphenyl borate.

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