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

Abstract

ICRAC (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 (ICRAC) 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.

Regulation of nuclear envelope permeability in cell death and survival

“…We also recorded capacitative Ca 2+ influx, a secondary Ca 2+ response ensuing in cells with depleted internal stores when Ca 2+ is added back to the culture medium. 49 In Bcl-2 overexpressing cells, this response was shown to be downregulated as adaptation to the long-term reduction of Ca 2+ in their internal stores. 50 Readdition of Ca 2+ (1 mM) to HeLa cells previously exposed to TG evoked a steep increase in fluorophore signal indicative of Ca 2+ capacitative influx.…”

Secretion of IL-2 and IFN-γ, But Not IL-4, by Antigen-Specific T Cells Requires Extracellular ATP

“…Because receptor-mediated activation of T cells and B cells is also dependent upon sustained influx of extracellular Ca 2ϩ (52-54), we asked whether ionomycin and PMA could overcome the ATP␥S-mediated inhibition of IL-2 secretion from the T cell hybridomas. Ionomycin induces capacitative calcium entry by depleting intracellular calcium stores (55,56), whereas PMA induces release of calcium from intracellular stores by activating protein kinase C or p21 ras (57). The requirement for both PMA and ionomycin to reverse the inhibitory effects of ATP␥S suggests that ecto-ATPase might be regulating TCRmediated intracellular signaling at two distinct steps.…”

Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

“…Indeed, ER depletion of comparable degree was reported to cause a substantial activation (over 50%) of store-dependent Ca 2 þ influx. 50 Conversely, Bcl-2 expression markedly reduces the [Ca 2 þ ] c increase induced by Ca 2 þ readdition to cells in which the Ca 2 þ store had been fully depleted by SERCA blockers. 37 This effect, that may represent a long-term adaptation to lower [Ca 2 þ ] er levels, is most likely due to reduction of the number of functional channels in the plasma membrane.…”