Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 9, Pages 3603-3608Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0609573104
Keywords
membrane protein; neurotransmitter : sodium symporter; scintillation proximity; substrate binding
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Funding
- NIDA NIH HHS [R01 DA017293, K05 DA022413, DA17293] Funding Source: Medline
- NIMH NIH HHS [K02 MH057324, MH57324] Funding Source: Medline
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Transport proteins constitute approximate to 10% 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.
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