Monitoring the function of membrane transport proteins in detergent-solubilized form

Abstract

Transport proteins constitute Ϸ10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment.membrane protein ͉ neurotransmitter:sodium symporter ͉ scintillation proximity ͉ substrate binding M embrane transport proteins fulfill an essential function in every living cell by catalyzing the translocation of solutes, including ions, nutrients, neurotransmitters, and numerous drugs, across biological membranes. Their (mal)function is directly implicated in many diseases including autism, epilepsy, migraine, depression, drug abuse, and cystic fibrosis, and they play an important role in the success or lack of success of cancer chemotherapy. Hence, they are of primary medical/pharmacological interest for target-oriented drug discovery and delivery. Their function is routinely studied in suitable expression hosts and, if feasible, after reconstitution into proteoliposomes. Direct biophysical and structural studies, including crystallization, however, require that these integral membrane proteins be solubilized and purified (Fig. 1). Between their extraction from the membrane and reconstitution, transport activity cannot be measured because of the lack of a vectorial environment. As a consequence, the determination of their function in detergent has been limited to indirect binding studies, such as substrate protection against cysteine modification (1-3) or substrate-induced changes in tryptophan fluorescence (3, 4), approaches that require the fortuitous or engineered localization of cysteines or tryptophans as well as laborious development and implementation.To overcome this significant bottleneck in membrane transporter proteomics, we developed a direct radiotracer-binding assay using scintillation proximity (5-8) to monitor the function of transport proteins in crude membrane extracts and in purified form. Because this enormous family of membrane transport proteins is still replete with many hypo...

Interaction of α-Lipoic Acid with the Human Na+/Multivitamin Transporter (hSMVT)

“…4). In this assay, purified recombinant hSMVT was immobilized on copper-coated scintillation beads (using the hexa-His tag in recombinant hSMVT) that emit light only when a radioactive ligand stably binds to the immobilized target protein (26). 0.83 M R- [ 3 H]LA yielded a robust specific SPA signal when 50 ng (about 0.73 pmol) of purified hSMVT was used per assay (Fig.…”

Chloride binding site of neurotransmitter sodium symporters

“…The structures were refined with PHENIX (56). Radiotracer binding studies were performed by means of the scintillation proximity assay (SPA) using copper-coated YSi SPA beads to capture the His-tagged LeuT variants (7,32). All experiments were repeated at least in duplicate.…”

Structure determination of channel and transport proteins by high-resolution microscopy techniques

“…A rapid and sensitive method to assay the substrate binding properties of mutants and of uncharacterized orphan target proteins in general is the scintillation proximity assay (SPA). In 2007, Matthias Quick (Quick and Javitch, 2007) introduced the application of this method to detergent-solubilized membrane transport proteins. We have established the SPA method for detergentsolubilized transporters in Bern, which is now being used routinely.…”