In situ Ca2+ titration in the fluorometric study of intracellular Ca2+ binding

Imaging with Ca2+-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca2+ signaling. The spatiotemporal evolution of intracellular free Ca2+ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca2+ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca2+ imaging data, and can be exploited to investigate endogenous Ca2+ buffer properties. Here we extended the use of Ca2+ fluorometry in the characterization of Ca2+ binding molecules within cells, building on a method of titration of intracellular Ca2+ binding sites in situ with measured amounts of Ca2+ entering through voltage-gated Ca2+ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca2+ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca2+ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca2+ buffer and dye, this method yielded estimates of the endogenous buffer concentration and Id, the dye Id, and the fraction of Ca2+ inaccessible cellular volume. The in situ Id of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca2+-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca2+ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca2+ binding species. This method offers a versatile approach to the study of endogenous Ca2+ binding molecules in their physiological milieu. (C) 2014 Elsevier Ltd. All rights reserved.