Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H + leak (I H ) across their inner membrane 1 . I H results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat 2-6 and ADP/ATP carrier (AAC) in other tissues 1,7-9 , but the underlying mechanism is poorly understood. As evidence of pharmacological activators of I H through UCP1 and AAC is lacking, I H is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) 10,11 . Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models 12-14 , their clinical potential for treating human disease is limited due to indiscriminately increasing H + conductance across all biological membranes 10,11 and adverse side effects 15 . Here we report the direct measurement of I H induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent I H through AAC. Thus, common protonophoric uncouplers are synthetic activators of I H through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.