Using two dyes to observe the competition of Ca2+ trapping mechanisms and their effect on intracellular Ca2+ signals.

The specificity and universality of intracellular [Formula: see text] signals rely on the variety of spatio-temporal patterns that the [Formula: see text] concentration can display. [Formula: see text] liberation through inositol 1,4,5-trisphosphate receptors ([Formula: see text]) is key for this variety. In this paper, we study how the competition between buffers of different kinetics affects [Formula: see text] signals that involve [Formula: see text] release through [Formula: see text]. The study also provides insight into the underlying spatial distribution of the channels that participate in the signals. Previous works on the effects of [Formula: see text] buffers have drawn conclusions 'indirectly' by observing the [Formula: see text]-bound dye distributions in the presence of varying concentrations of exogenous buffers and using simulations to interpret the results. In this paper, we make visible the invisible by observing the signals simultaneously with two dyes, [Formula: see text] and [Formula: see text], each of which plays the role of a slow or fast [Formula: see text] buffer, respectively. Our observations obtained for different concentrations of [Formula: see text] highlight the dual role that fast buffers exert on the dynamics, either reducing the intracluster channel coupling or preventing channel inhibition and allowing the occurrence of relatively long cycles of [Formula: see text] release. Our experiments also show that signals with relatively high [Formula: see text] release rates remain localized in the presence of large [Formula: see text] concentrations, while the mean speed of the elicited waves increases. We interpret this as a consequence of the more effective uncoupling between [Formula: see text] clusters as the slow dye concentration increases. Combining the analysis of the experiments with numerical simulations, we also conclude that [Formula: see text] release not only occurs within the close vicinity of the centers of the clearly identifiable release sites ([Formula: see text] clusters) but there are also functional [Formula: see text] in between them.