Project Summary Mitochondria control cell metabolism by converting nutrients into an electrochemical gradient of protons (H+) across the inner mitochondrial membrane (IMM) to generate ATP, the currency of the cell, and heat (called mitochondrial thermogenesis). A precise balance in the distribution of H+ between the two forms of energy production, ATP and heat, defines the metabolic homeostasis of the cell. Brown fat and beige fat mitochondria specialize in the production of heat via the uncoupling protein 1 (UCP1). However, even in other tissues, mitochondrial thermogenesis accounts for 25% of total mitochondrial energy production and can therefore have a considerable impact on the physiology of the entire body. Mitochondrial thermogenesis is not only essential for maintaining core body temperature, it is also the process by which excess calories are burned to prevent diet- induced obesity. In addition, it reduces the production of reactive oxygen species (ROS) by the mitochondria to protect cells from oxidative damage. In addition, chemical uncouplers such as 2,4-dinitrophenol (DNP), which are believed to increase H+ leak independently of proteins, are the most effective anti-obesity drugs to date. Thus, mitochondrial thermogenesis is a powerful regulator of cellular metabolism, and a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction, including metabolic syndrome and age-related disorders. Unfortunately, the precise molecular mechanisms that control the acute activation of thermogenesis in the mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for direct measurement of H+ currents across the IMM. The development of a methodology based on the patch-clamp technique allows for the first time the direct study of H+ leak through the IMM of each tissue and the first biophysical characterization of mitochondrial transporters, such as UCP1 and the ADP/ATP transporter (AAC), which are the mediators of this H+ leak. This unique approach now provides an unprecedented high- resolution direct functional analysis of 1) the mitochondrial ion channels and transporters responsible for mitochondrial thermogenesis and 2) the mechanisms of action of chemical uncouplers such as DNP. Using the new mitochondrial patch-clamp assay combined with modern cellular and molecular techniques, this research project will provide new insights into the mechanisms that control the thermogenic capacity of the mitochondria and how they can be targeted for therapeutic purposes.