Electrophysiological Characterization of Mitochondrial Uncoupling Protein 1

Abstract

Uncoupling proteins (UCP1- UCP5) are integral transport proteins of the inner mitochondrial membrane (IMM). They mediate transmembrane ion leak, thus dissipating the electrochemical proton gradient across the IMM and uncoupling mitochondrial respiration and ATP synthesis. UCPs are involved in thermogenesis, reducing fat deposition, and attenuating reactive oxygen species production by mitochondria to protect the cell against oxidative damage and ageing. The mechanism of ion conductance of UCPs has long remained elusive due to the lack of direct methods for measuring ion currents produced by them. To resolve this problem, we applied the patch-clamp technique to the whole inner membrane of mitochondria from the brown adipose tissue to identify and characterize currents produced by the family's founding member, UCP1. In our experiments, both the cytoplasmic and matrix faces of the IMM were exposed to solutions that did not contain any ions normally permeable through ion channels or transporters, except for H+ and OH-. Under these conditions, we identified a current that showed all signature properties of UCP1: it was strongly potentiated by micromolar concentrations of unsaturated fatty acids, inhibited by removing endogenous membrane fatty acids with bovine serum albumin, and blocked by micromolar concentrations of purine nucleotides. Moreover, the current was absent in the IMM of the kidney COS-7 cells, in agreement with the fact that UCP1 is specifically expressed in the brown adipose tissue. The transport molecule mediating this current was activated by membrane depolarization and showed robust tail currents upon return to the negative potentials. In a symmetrical pH 5.0 solution the current amplitude was negligible as compared to a symmetrical pH 8.0 solution, suggesting that the current was carried by OH- but not H+. Our results indicate that UCP1 is a low-conductance OH- channel weakly activated by membrane depolarization.