Study of the Capacity of Each IP3 Receptor Isoform to Support ER-Mitochondrial Calcium Transfer

Abstract

IP3 receptors (IP3R) relay calcium signals locally from the ER to the mitochondria, which process is central to the control of oxidative metabolism and to the induction of mitochondrial apoptosis. IP3R have 3 isoforms that show heterogeneous structure, function and intracellular distribution. Both IP3R1 and IP3R3 have been linked to local calcium transfer between the ER and mitochondria but systematic evaluation of each isoform in the same paradigm has not been conducted. To fill this void we used IP3R-deficient DT40 chicken B cells stably rescued with each mammalian IP3R isoform biochemically tagged to allow detection by a single antibody. Immunofluorescence validated clones with similar expression level of each isoform. In these clones, fluorometric measurements showed comparable calcium pool sizes for all isoforms but different IP3 sensitivities (IP3R2≥IP3R1>>IP3R3). In simultaneous single cell fluorescence imaging of cytoplasmic and mitochondrial matrix [Ca2+], all three isoforms supported propagation of calcium signals to the mitochondria upon stimulation with trypsin but with different efficacy and kinetics. We are applying fluorescence-based microscopy and transmission electron microscopy to study the topology of the ER-mitochondrial interface and the localization of IP3R at the ER-mitochondrial interface. Our results describe a paradigm that allows side-by-side comparison of each IP3R isoform in terms of ER-mitochondrial local calcium transfer and are expected to reveal the relevance of localization and other factors including Ca2+ flux kinetics for effective interorganellar Ca2+ transfer.