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.