CAPACITATIVE CA2- A STUDY USING SIMULTANEOUS MEASUREMENTS OF I-CRAC AND INTRALUMINAL [CA2+]( ENTRY IS CLOSELY LINKED TO THE FILLING STATE OF INTERNAL CA2+ STORES )

I-CRAC (the best characterized Ca2+ current activated by store depleti on) was monitored concurrently for the first time with [Ca2+] changes in internal stores, To establish the quantitative and kinetic relation ship 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-affinit y 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 re duction of free [Ca2+] within the ER and (b) by any measurable decreas e in [Ca2+](ER). We also demonstrate that the intrinsic kinetics of in activation are relatively slow and possibly dependent on soluble facto rs that are lost during the whole-cell recording.