Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Abstract

Mitochondria generate heat due to H+ leak (IH) across their inner membrane. IH results from the action of long-chain fatty acids (FA) on uncoupling protein 1 (UCP1) in brown fat and ADP/ATP carrier (AAC) in other tissues, but the underlying mechanism is poorly understood. Because no pharmacological activators of IH via UCP1 and AAC are known, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) that are thought to increase IH independently of membrane proteins. Protonophores are highly effective in combating obesity, insulin resistance, type II diabetes, and fatty liver in animal models, but because they indiscriminately increase H+ conductance across all biological membranes and have adverse side effects, their use in humans is restricted. Here we report the first direct measurement of IH induced by common protonophores and discover that their mechanism of action in mitochondria was significantly misunderstood. IH induced by DNP and other protonophores depends on AAC and UCP11. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain FA 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 IH via AAC. Thus, common protonophoric uncouplers emerge as the first synthetic activators of IH via UCP1 and AAC identified to date, paving the way for the development of new and more specific activators of these two proteins.