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

Research paper by Estefania E Piegari, Lucia Fernanda LF Lopez, Silvina S Ponce Dawson

Indexed on: 01 Jun '18Published on: 01 Jun '18Published in: Physical biology


The specificity and universality of intracellular Ca<sup>2+</sup> signals rely on the variety of spatio-temporal patterns that the Ca<sup>2+</sup> concentration can display. Ca<sup>2+</sup> liberation through inositol 1,4,5-trisphosphate receptors (IP<sub>3</sub>Rs) is key for this variety. In this paper we study how the competition between buffers of different kinetics affects Ca<sup>2+</sup> signals that involve Ca<sup>2+</sup> release through IP<sub>3</sub>Rs. The study also gives insight on the underlying spatial distribution of the channels that participate of the signals. Previous works on the effects of Ca<sup>2+</sup> buffers drew conclusions ``indirectly'' by observing the Ca<sup>2+</sup> -bound dye distributions in the presence of varying concentrations of exogenous buffers and using simulations to interpret the results. In this paper we {\it make visible the invisible} by observing the signals simultaneously with two dyes, Rhhod-2 and Fluo-4, each of which plays the role of a slow or a fast Ca<sup>2+</sup> buffer, respectively. Our observations obtained for different concentrations of Fluo-4 highlight the dual role that fast buffers exert on the dynamics, either reducing the intracluster channel coupling or preventing the channels inhibition and allowing the occurrence of relatively long cycles of Ca<sup>2+</sup> release. Our experiments also show that signals with relatively high Ca<sup>2+</sup> release rates remain localized in the presence of large Rhod-2 concentrations while the mean speed of the elicited waves increases. We interpret this as a consequence of the more effective uncoupling between IP<sub>3</sub>R clusters as the slow dye concentration increases. Combining the analysis of the experiments with numerical simulations we also conclude that Ca<sup>2+</sup> release not only occurs within the close vicinity of the centers of the clearly identifiable release sites IP<sub>3</sub>R clusters) but there are also functional IP<sub>3</sub>Rs in between them. © 2018 IOP Publishing Ltd.