On the Role of Positively Charged Residues of TM2 Domain in the Chloride Transport of Human UCP2

Abstract

Located in the inner mitochondrial membrane, uncoupling proteins (UCPs) dissipate the proton electrochemical gradient causing reduction in the rate of ATP synthesis. Among five human UCP homologues, UCP2 is unique with its ubiquitous expression in various tissues. This important feature has been attributed to UCP2's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, glucose and lipid metabolisms. Despite numerous physiological studies, UCP2 function in cell remains poorly understood. UCP2 proton transport is regulated by purine nucleotides such as ATP, ADP, GTP and GDP. In addition, UCP2 has also been observed to transport chlorides and other small anions. Identification of the key amino acid residues in UCP2 in proton, anion transport and regulation will help determine the protein's mechanism of action in cells. It has been established that positively charged residues on transmembrane helix 2 (TM2) of UCP1 and UCP2 are crucial for chloride transport. However, a full understanding of the transport mechanism is yet to be achieved. More importantly, some of these residues are also involved in the UCP2 proton transport regulation. To further understand the ion transport of UCP2, four TM2 mutants have been made (R76Q, R88Q, R96Q, and K104Q). Over-expressed proteins were purified and reconstituted into liposomes for structural and functional studies. All mutants share an overall helical conformation with wtUCP2. using anion-sensitive fluorescent probes, proton and chloride transport of UCP2 mutants are examined to determine the effect of each mutation on the ion transport of UCP2. In addition, Mant-modified purine nucleotide will be used to study the binding of UCP2 and its mutants to purine nucleotides. Overall, the outcome of this study will provide a more detailed molecular image of UCP2 ion transport mechanism.