Molecular Biophysics of Mitochondrial Membranes: Defining Future Therapeutic Targets Summary Mitochondrial dysfunction is implicated in several devastating diseases, such as neurodegeneration, obesity, diabetes, and cancer. Pharmacological interventions at the level of mitochondria can become an effective way to treat these pathological conditions. However, the development of such therapeutic tools is prevented by our incomplete understanding of the molecular mechanisms that underlie major mitochondrial functions, including energy production, setting the pace of aging, and controlling cell death. The transport of ions and molecules across the mitochondrial membranes is the foundation of the mitochondrial physiology and a lack of direct methods to study mitochondrial transmembrane transport is likely the most significant barrier to a better understanding of mitochondria. The key mitochondrial transport proteins, such as ATP synthase, the electron transport chain, and ion channels of the inner and outer mitochondrial membranes, could be best studied using the patch-clamp technique. This method revolutionized our understanding of ion channels and electrogenic transporters of the plasma membrane; however, an analogous application of the patch-clamp technique to mitochondria has been extremely difficult due to their small size and double- membrane architecture. Here we propose to develop an easily reproducible method for the application of the patch-clamp technique to both the inner and outer mitochondrial membranes for routine use in mitochondrial research. We will then apply the whole-membrane and single-channel modes of the patch- clamp technique to identify the full complement of ion channels and electrogenic transporters that are present in the inner and outer mitochondrial membranes. The accomplishment of these aims will provide an unparalleled functional essay for the key mitochondrial transport proteins, which, when combined with molecular biology, genetics, and protein crystallography, will facilitate significant advances in our understanding of the molecular workings of mitochondria and the subsequent development of therapeutic tools that control mitochondrial functions.