Calcium-Induced Redox Microdimains at the ER-Mitochondrial Interface

Abstract

Diverse interactions between Ca2+ and reactive oxygen species (ROS) signalling are thought to exist and be of both physiological and pathophysiological relevance. We hypothesized that ROS and Ca2+ interact locally at the ER-mitochondrial interface to induce mitochondrial Ca2+-overload and subsequent permeability transition. To test the relationship between ROS and Ca2+, we used genetically-encoded tools to selectively generate and measure ROS at confined locations within HepG2 and MEF cells. Using Killer Red (KR) targeted to the ER-mitochondrial interface or nucleus, we selectively controled the oxidation of these compartments with illumination. When KR was targetted to the ER-Mitochondrial interface, illumination caused a >3-fold increase in cytoplasmic Ca2+ response to a low-dose IP3-linked agonist as measured with fura 2, whereas illumination of the KR targeted to the nucleus was ineffective. These data demonstrate that ROS generated at the ER-mitochondrial interface are capable of sensitizing Ca2+ release, whereas more distant ROS was not. To test if (patho)physiological conditioins could oxidise the ER-mitochondrial interface in the same way, we utilized the genetically-encoded ratiometric sensor HyPer and the redox de-sensitized derivative SypHer to control for non-specific changes. These probes, targeted to the ER-mitochondrial interface or nucleus, are positioned to assess redox changes at a local level. When permeabilized MEF cells were subject to mitochondrial Ca2+ overload, a cyclosporine A-sensitive decrese in mitochondrial membrane potential, consistent with permeability transition, was observed. This was accompanied by a pronounced burst of H2O2 at the ER-mitochondrial interface but not the nucleus. These data demonstrate that the ER-mitocondrial interface may host a local redox microdomain and that ROS generated at locations immediately apposed to Ca2+-transport proteins, such as the IP3 receptor, sensitize Ca2+ release whereas distant ROS did not. These observations underline the critical role of spatial organization in ROS-Ca2+ crosstalk in mitochondrial signalling.