Mitochondrial uncouplers induce proton leak by activating AAC and UCP1

Mitochondria generate heat due to H⁺ leak (I_H) across their inner membrane¹. 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 effects¹⁵. 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.