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

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