SCREENING LIGANDS FOR MEMBRANE-PROTEIN RECEPTORS BY TOTAL INTERNAL-REFLECTION FLUORESCENCE - THE 5-HT3 SEROTONIN RECEPTOR

The screening of ligands for membrane receptor proteins is central to the discovery of new pharmaceutical drugs. We present a general method to reversibly attach receptor proteins via an affinity tag to a quart z surface and subsequently detect with high sensitivity the real-time binding of ligands by total internal reflection fluorescence. A seroto nin-gated ion channel protein was immobilized, and the binding of a fl uorescent ligand was investigated. The affinity and the kinetic parame ters of binding were measured, and the effect of unlabeled compounds w as determined by competition. The pharmacology of the immobilized rece ptor was identical to that of the native receptor. The affinity of unl abeled ligands was rapidly and effectively determined. The method desc ribed here is generally applicable for membrane proteins and opens new ways for the discovery of pharmacologically active compounds.