Conformation and Ion Transport of Neuronal Uncoupling Proteins

Abstract

Located in the inner mitochondrial membrane, uncoupling proteins (UCPs) dissipate the proton electrochemical gradient causing reduction in the rate of ATP synthesis. Three UCP homologues (UCP2, UCP4 and UCP5), expressed in neurons, are suggested to have potential roles in the function and protection of the central nervous system (CNS). Extensive biochemical studies on UCP2 have provided adequate evidence for its participation in proton and anion transport. So far, no functional studies in proteoliposome systems have been performed on UCP4 and UCP5. Due to the lack of high resolution structures, structural information for UCPs remains limited. The goals of this study are to gain further information on the conformations and functional properties of neuronal UCPs reconstituted in liposomes. The emphasis is on UCP4 and UCP5 and their comparison to UCP2. Recombinant versions of all five UCPs have been successfully expressed, purified and reconstituted in liposomes. All UCPs formed dominantly helical conformations in negatively charged phospholipid vesicles as detected by CD spectroscopy [Ivanova et al (2010) Biochemistry 49(3): 512-21]. Neuronal UCPs also share common structural and binding properties with UCPs 1 and 3, implying a common physiological role in addition to their specific roles in the CNS. Ion transport assays (proton and chloride) for reconstituted neuronal UCPs have been developed using anion-sensitive fluorescent probes. Purine nucleotide inhibitions of these proteins resulted in decrease in the initial transport rate. Preliminary results show that reconstituted UCP5 has proton transport activity that is inhibited by ATP. Similar experiments are planned for UCP4. Transport activities of the all neuronal UCPs will be compared to those of the prototypical UCP1. The outcome of this study will clarify several aspects of the structure-function relationships of the least studied UCPs 4 and 5 and their possible physiological roles in the CNS.