Calcium Buffering by Fluorescent Indicators - Implications and Easy Solutions

Abstract

Fluorescent indicators are powerful tools to determine intracellular free Ca2+ concentrations ([Ca2+]). Although their properties vary widely, indicators share common design and operation principles. Being composed of a Ca2+ binding moiety, usually BAPTA-derived, and a fluorophore such as fluorescein, an indicator must bind calcium ions to change its fluorescence. Therefore, within cells, indicators compete with the endogenous buffers for calcium and act as additional buffers, which, in turn, can thwart comparisons of indicator responses and complicate estimating free [Ca2+] within the cell ([Ca2+]in). As lower indicator concentrations tend to report higher [Ca2+], we hypothesized that the true (i.e. indicator-free) [Ca2+]in could be determined by extrapolating the reported [Ca2+]in to the indicator free environment. Although conceptually similar to predecessors, we present a steady state method that avoids the complexities of earlier approaches, and requires only data collected in AM ester-loaded cells. As such, it can be applied to determine the indicator-independent peak [Ca2+]in that is induced by any stimulus in any cell type. To test this approach, we used fura-2 in well-defined in vitro systems and found that it estimated not only [Ca2+]in, but also, albeit less accurately, the concentration and affinity of endogenous buffers. When applied to data collected in fura-2 loaded neurons depolarized with 50 mM K+, the method estimated an indicator independent [Ca2+]in in resting (22 nM) and stimulated (1.18 µM) neurons and characterized the endogenous buffer (∼600 µM) as having relatively low affinity (0.78≤Kb≤1.63 µM), results close to those reported previously. While the method does not account for indicator-distorted Ca2+ decay rates (a consequence of omitting the time component), it does create a simple and general way to fix peak [Ca2+] amplitudes, the values that are most often measured and compared in routine Ca2+ imaging experiments.