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

PIEGARI, ESTEFANIA; LOPEZ, LUCIA FERNANDA; PONCE DAWSON, SILVINA

Physical Biology

IOP Publishing

Año: 2018

The specificity and universality of intracellular Ca2+ signals rely on the varietyof spatio-temporal patterns that the Ca2+ concentration can display.Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) is keyfor this variety. In this paper we study how the competition between buffersof different kinetics affects Ca2+ signals that involve Ca2+ release throughIP3Rs. The study also gives insight on the underlying spatial distribution ofthe channels that participate of the signals. Previous works on the effects ofCa2+ buffers drew conclusions ?indirectly? by observing the Ca2+-bound dyedistributions in the presence of varying concentrations of exogenous buffersand using simulations to interpret the results. In this paper we make visiblethe invisible by observing the signals simultaneously with two dyes, Rhod-2and Fluo-4, each of which plays the role of a slow or a fast Ca2+ buffer, respectively.Our observations obtained for different concentrations of Fluo-4highlight the dual role that fast buffers exert on the dynamics, either reducingthe intracluster channel coupling or preventing the channels inhibitionand allowing the occurrence of relatively long cycles of Ca2+ release. Ourexperiments also show that signals with relatively high Ca2+ release rates remainlocalized in the presence of large Rhod-2 concentrations while the meanspeed of the elicited waves increases. We interpret this as a consequence ofthe more effective uncoupling between IP3R clusters as the slow dye concentrationincreases. Combining the analysis of the experiments with numericalsimulations we also conclude that Ca2+ release not only occurs within theclose vicinity of the centers of the clearly identifiable release sites (IP3Rclusters) but there are also functional IP3Rs in between them.