Abstract
Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory signaling in the central nervous system. When a ligand binds to the extracellular domain of iGluRs, local conformational changes ensue and this motion is translated to the transmembrane domain, inducing channel opening. We have used an isolated ligand binding domain, GluR2-S1S2J (GluR2), as a model system to study the protein-ligand complex by steady-state and time-resolved intrinsic tryptophan fluorescence measurements. Specifically, we determined that the widely used and structurally characterized antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), acts as an efficient fluorescence energy transfer (FET) acceptor for Trp. Consistent with crystallographic data, our results indicate that the four native tryptophans are within Forster's radius (R(o) approximately 33 A) of the bound ligand. Additionally, we demonstrate the broader value of this technique by identifying an original FET ligand, 3-nitrotyrosine (3NY), for GluR2 (R(o) approximately 24 A; apparent dissociation constant K(d) approximately 170 microM). Estimated average donor-acceptor (Trp-ligand) distances extracted from tryptophan excited-state decays are similar for both ligands (approximately 24 A), suggesting that 3NY binds in the structurally characterized ligand binding cleft. Moreover, an alternative competition assay utilizing Trp --> DNQX quenching for detection of ligand binding in GluR2 is described.