An improved measurement of the Ca²⁺-binding affinity of fluorescent Ca²⁺ indicators

Fluorescent Ca²⁺ indicators are widely used to measure the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in living cells, including neurons. By calibrating an indicator in solutions that mimic the main ionic constituents of the actual cytoplasm, [Ca²⁺]_i can be determined from the measured fluorescence intensity. However, different studies have reported considerably different Ca²⁺-binding affinities (K_d) for the same indicator, even though they used calibrating solutions with similar compositions. In this paper, we present a method to accurately determine the K_d values of non-ratiometric Ca²⁺ indicators in solutions that mimicked a standard patch-clamp internal solution. The free Ca²⁺ concentration ([Ca²⁺]) in these solutions, which was set by either EGTA or HEDTA, was measured with a Ca²⁺-selective macroelectrode. We found that such a measurement was critical for an accurate calibration of the Ca²⁺ indicators. The K_d values of OGB-1, OGB-6F, fluo-5F, and fluo-4FF were 0.26 ± 0.01, 8.7 ± 0.4, 1.00 ± 0.05, and 23.0 ± 0.7 μM, respectively. Calculating [Ca²⁺] with Maxchelator, a widely used computer program, led to a significant underestimation of the K_d values of OGB-6F, fluo-5F, and fluo-4FF. This is because the purity of EGTA was considerably less than that advertised by the manufacturer. In addition, the K_d value of HEDTA was overestimated by Maxchelator. Therefore, besides batch-to-batch variations, the fact that [Ca²⁺] in the calibrating solutions of many studies was estimated with Maxchelator is very likely a reason for the different published values of K_d of Ca²⁺ indicators. Using a reaction-diffusion model to reproduce Ca²⁺ rises in a nerve terminal, we further showed that incorrect calibration of fluorescent Ca²⁺ indicators can underlie the large variation of the endogenous Ca²⁺ binding ratio between different types of excitatory synapses.