Capacitative Ca²⁺ Entry Is Closely Linked to the Filling State of Internal Ca²⁺ Stores: A Study Using Simultaneous Measurements of I_CRAC and Intraluminal [Ca²⁺]

I_CRAC (the best characterized Ca²⁺ current activated by store depletion) was monitored concurrently for the first time with [Ca²⁺] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca²⁺] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca²⁺ chelator N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca²⁺] within the ER can be lowered and restored to its original level within 10–15 s without modifications of Ca²⁺ pumps or release channels. Using these new tools, we demonstrate here that Ca²⁺ release–activated Ca²⁺ current (I_CRAC) is activated (a) solely by reduction of free [Ca²⁺] within the ER and (b) by any measurable decrease in [Ca²⁺]_ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.