A FLUOROMETRIC METHOD FOR ESTIMATING THE CALCIUM CONTENT OF INTERNAL STORES

The concentration of Ca2+ in intracellular stores is an important fact or in many aspects of Ca2+ signaling, including the generation of Ca2 spikes, oscillations and waves, control of mitochondrial respiration, and activation of store-operated Ca2+-channels. Here we describe a co nsistent method for estimating the content of stores, based on the rel ease of stored Ca2+ by thapsigargin (TG) or ionomycin (IO). Once relea sed from stores, Ca2+ elevates [Ca2+](i) transiently before it is pump ed across the plasma membrane. If the dependence of the pump rate on [ Ca2+](i) is known, then the kinetics and amplitude of the Ca2+ transie nt allows the total amount of releasable Ca2+ to be estimated. We deve lop this quantitative approach and validate its use in human T cells, in which the Ca2+ clearance rate is an approximately linear function o f [Ca2+](i). Our results support the assumption that the ER Ca2+ leak in resting T cells is unregulated, i.e. its rate is proportional to lu minal [Ca2+]. The characteristic time constant for basal Ca2+ release is 110-140 s, comparable to that for activation of Ca2+ release-activa ted Ca2+ (CRAC) channels by TG and consistent with the dependence of I -CRAC on store depletion. This method for estimating store content may be useful for quantifying the overlap between functionally distinct s tores and for defining the relation between store content and cellular responses.