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

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.

Diffusion influenced binary reactive processes in membranes involving identical particles: a Monte Carlo study.

Simple random walk simulations on triangular lattices were performed in order to obtain a basic quantitative understanding of the kinetics of diffusion influenced binary reactive processes of membrane associated peptides or proteins within the two dimensionality of lipid bilayers. The results of the Monte Carlo simulations are compared with various formal approximate steady-state approaches, such as presented by Keizer [Acc. Chem. Res., 18 (1985) 235-241] in the context of statistical nonequilibrium thermodynamics or by Hardt [Biophys. Chem., 10 (1979) 239-243], based on the well known work of Delbrück and Adam. For diffusion controlled binary reactions of identical particles, nice agreement with the numerically simulated values is found in the low concentration limit for both Hardt's and Keizer's approach. For the latter a fluctuating steady-state particle source has to be considered. The dependence of the steady-state rate coefficient on system size is investigated, and the results are compared to the work of Swartz and Peacock-López [J. Chem. Phys., 95 (4) (1991) 2727-2731]. In order to elucidate the results, a practical application is considered. An application to a dimerization reaction on vesicles of typical experimental dimensions is given.