IP3 Receptor Isoform Dependence of the ER-Mitochondrial CA2+-Transfer in Mammalian Cells

Abstract

IP3 receptors (IP3Rs) mediate Ca2+ release from the ER to the cytosol and can relay Ca2+ signals locally to mitochondria. The IP3R family is composed by three isoforms (type 1,2 and 3) showing heterogeneous structure, function and tissue distribution. In some paradigms specific IP3R isoforms were shown to be enriched close to and support Ca2+ transfer locally to the mitochondria. Until recently, a mammalian system allowing systematic evaluation of each isoform has remained unavailable. The establishment of IP3R triple knock out (TKO) HEK cells and rescue with different IP3R isoforms tagged with a common biochemical tag provided new opportunities to test the hypothesis that IP3R isoforms are equal in terms of coupling to the mitochondria. To study Ca2+ coupling between ER and mitochondria we used WT HEK cells and TKO cells acutely and stably rescued by the different isoforms of IP3Rs. Ca2+-signaling was induced by an IP3-generating agonist, carbachol. To measure changes in mitochondrial Ca2+ ([Ca2+]m) we transfected cells with the genetically-encoded Ca2+ sensor CEPIA targeted to the mitochondrial matrix (mtCEPIA), changes in cytosolic Ca2+ ([Ca2+]c) were measured by FURA-2 AM simultaneously. WT HEK cells showed a marked increase in [Ca2+]c followed by a fast rise in [Ca2+]m with the coupling time of 0.94 ±0.22 s (n=56) while TKO cells showed no changes in [Ca2+]c and [Ca2+]m to agonist stimulation. Acute and stable type 1, 2 or 3 IP3R rescue TKO cells regained [Ca2+]c responses and also showed a [Ca2+]m increase. Quantitative analysis of the Ca2+ transfer efficiency to the mitochondria and the localization of each IP3R isoform relative to the mitochondria are currently pursued. In summary, we have validated a new model for the study of IP3R-mediated local Ca2+ signaling between the ER and mitochondria, allowing us to determine the specific function of each isoform in these processes.