Liposome reconstitution and transport assay for recombinant transporters

Abstract

Secondary active transporters are responsible for the cellular uptake of many biologically important molecules, including neurotransmitters, nutrients, and drugs. Because of their physiological and clinical importance, a method for assessing their transport activity in vitro is necessary to gain a better understanding of how these transporters function at the molecular level. In this chapter, we describe a protocol for reconstituting the concentrative nucleoside transporter from Vibrio cholerae into proteoliposomes. We then describe a radiolabeled substrate uptake assay that can be used to functionally characterize the transporter. These methods are relatively common and can be applied to other secondary active transporters, with or without some modification.

Keywords: Concentrative nucleoside transporter; Lipid vesicle; Proteoliposome; Radioactive uptake assay; Reconstitution; Sodium-coupled; Transport assay; Transporter; vcCNT.