A fluorometric method for estimating the calcium content of internal stores.

摘要

The concentration of Ca2+ in intracellular stores is an important factor 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 consistent method for estimating the content of stores, based on the release of stored Ca2+ by thapsigargin (TG) or ionomycin (IO). Once released from stores, Ca2+ elevates [Ca2+]i transiently before it is pumped across the plasma membrane. If the dependence of the pump rate on [Ca2+]i is known, then the kinetics and amplitude of the Ca2+ transient allows the total amount of releasable Ca2+ to be estimated. We develop this quantitative approach and validate its use in human T cells, in which the Ca2+ clearance rate is an approximately linear function of [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 luminal [Ca2+]. The characteristic time constant for basal Ca2+ release is 110-140 s, comparable to that for activation of Ca2+ release-activated Ca2+ (CRAC) channels by TG and consistent with the dependence of ICRAC on store depletion. This method for estimating store content may be useful for quantifying the overlap between functionally distinct stores and for defining the relation between store content and cellular responses.