Fig. 5. Cartoon summary of mitochondrial role in I_CRAC in weak (physiological) intracellular Ca²⁺ buffer. (A) Shows the resting state, where I_CRAC is not functioning. Stores are full and any Ca²⁺ that leaks from the stores is re-sequestrated by the SERCA pumps. (B) Following an increase in the levels of the second messenger InsP₃ in the absence of active mitochondrial Ca²⁺ uptake, Ca²⁺ is released from the stores. However, the SERCA pumps are able to re-sequestrate sufficient Ca²⁺ to prevent the threshold for macroscopic activation of I_CRAC from being reached. Only a very small fraction of CRAC channels are activated (undetectable in whole-cell mode). Furthermore, the rise in cytosolic Ca²⁺ results in strong inactivation of I_CRAC through Ca²⁺-dependent slow inactivation. (C) In the presence of respiring mitochondria, InsP₃ activates macroscopic I_CRAC. Ca²⁺ released from the stores by InsP₃ is taken up by mitochondria through a ruthenium red-sensitive uniporter. This reduces the amount of Ca²⁺ available to the SERCA pumps and in the vicinity of open InsP₃ receptors, such that the stores are depleted sufficiently for macroscopic I_CRAC to activate (less refilling by SERCA pumps and less inactivation of InsP₃ receptors). I_CRAC now activates. Some refilling does occur because inclusion of thapsigargin enhances the size of the current. Furthermore, mitochondrial Ca²⁺ buffering reduces the rate and extent of Ca²⁺-dependent slow inactivation, thereby increasing the size and duration of the current. (D) A simplified gating scheme for CRAC channels summarizing the role of mitochondrial Ca²⁺ buffering. Mitochondria facilitate opening (Closed to Open transition) whilst simultaneously reducing inactivation (Open to Inactivated transition). In this way, mitochondria have a much larger impact on I_CRAC than through either transition alone.