Evaluating the role of luminal calcium on intracellular calcium signals mediated by IP3 receptors.

LUCIA LOPEZ, LORENA SIGAUT AND SILVINA PONCE DAWSON

Simposio; Yeast Systems Biology International Symposium; 2011

Institus Pasteur de Montevideo

Calcium signals are used by almost all cell types to initiate a large variety of processes. Calcium release through IP3 receptors (IP3R’s) is a key component in many of these signals. IP3R’s are located on the membrane of the endoplasmic reticulum, usually forming clusters. The channel becomes open upon binding IP3 and calcium to a cytosolic site. There are also calcium binding sites on the luminal side of the reticulum. However, the way that luminal calcium regulates the kinetics of IP3R’s or the role it plays on the dynamics of the signals are unknown. In this work we have taken the first steps towards an evaluation of this role. To this end, we have elaborated a protocol to load the reticulum of Xenopus Laevis oocytes with a calcium dye. We show the success of the protocol by observing simultaneously the lumen (marking calcium) and the membrane of the ER (using a specific marker). We then show IP3 mediated signals as observed from the lumen and from the cytosol simultaneously using two dyes that are differentially contained in both regions and that fluoresce with different wavelengths. This opens up the possibility of getting a quantitative understanding of luminal calcium on IP3 mediated calcium signals.