Immobilized P2X 2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening

Abstract

The purinergic receptor signaling system plays an important role in communication between cells in the nervous system and opens new opportunities for screening of potential drugs. Our objective was to explore the pharmacological properties and establish a new methodology for ligand screening for the P2X 2 receptor, which has been developed by the combinatorial library approach Systematic Evolution of Ligands by Exponential enrichment (SELEX). To this end, membranes of 1321N1 cells stably transfected with rat P2X 2 receptors were resuspended in 2% cholate detergent and subsequently coupled onto an immobilized artificial membrane (IAM). The IAM–cholate–P2X 2 mixture was then dialyzed, centrifuged and packed into a FPLC column. Equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2′3′- O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using 32P alpha ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [C.A. Trujillo, A.A. Nery, A.H. Martins, P. Majumder, F.A. Gonzalez, H. Ulrich, Biochemistry 45 (2006) 224–233]. In addition, we demonstrate that this assay can be used in in vitro selection procedures for RNA aptamers binding to P2X 2 receptors. The results demonstrate that the receptor can be immobilized in a stable format and reused over an extended period of time, facilitating the exploration of ligand–receptor interactions and screening of combinatorial pools for possible ligands.