SUMMARY Modulation of the cytoplasmic concentration of Ca2+ ([Ca2+]i) by inositol trisphosphate (InsP3)-triggered release of Ca2+ from the endoplasmic reticulum (ER) is a ubiquitous signaling system that regulates numerous cell physiological processes. InsP3-mediated [Ca2+]i signals are manifested as repetitive spikes or oscillations, and they can be highly localized or propagate to provide signals to discrete parts of the cell. At the heart of this complex signaling system is the InsP3R ion channel. We have provided rigorous understanding of the ion- channel properties of the InsP3R, by studying the channel using powerful quantitative single-channel patch- clamp electrophysiology of native ER membranes, a technique that we pioneered; how those properties are regulated by physiological agonists and protein interactions; and how changes in these properties are reflected in physiological outcomes. An important physiological target of InsP3R-mediated Ca2+ signals are mitochondria. InsP3R channels play a fundamental role in the regulation of cell metabolism, primarily by supplying released Ca2+ to mitochondria to stimulate TCA-cycle dehydrogenases to promote oxidative phosphorylation (OXPHOS) and ATP production. We discovered that low-level constitutive InsP3R-mediated Ca2+ release to mitochondria is essential for maintaining basal levels of OXPHOS and ATP production in most cell types, and that cancer cells have a particular reliance on this pathway for their survival. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial Ca2+ uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane (IMM). As for the InsP3R, we have employed biochemical and powerful biophysical approaches to understand the ion-channel properties of MCU, including patch-clamp electrophysiology of MCU Ca2+ currents in individual mitoplasts. Our overarching effort has been to quantitatively understand the molecular physiologies of the InsP3R and MCU channels whose integrated activities control cellular physiology and life and death decisions. Recently, cryo-electron microscopic (cryo-EM) structures of both the InsP3R and MCU have been solved. Because of our exertise in the biophysics and molecular physiology of these intracellular ion channels, we are uniquely positioned to exploit this new information to address important questions regarding the molecular mechanisms of ion permeation and channel gating and their regulation of both Ca2+ ion channels. Our goals are to understanding the molecular mechanisms of InsP3R channel gating regulation, to gain fundamental new insights into the molecular mechanisms of MCU channel ion permeation and gating regulation, including by interacting mitochondrial proteins, and to exploit the information gained from the first two goals to provide quantitative insights into ER-to-mitochondrial Ca2+ transfer. Because of the fundamental reliance of cancer cells on this signaling system and its role in familial Alzheim...