A Mutational Analysis Probing the Structural Features Underlying Ligand Binding in Kainate Receptors:
Similarities to Bacterial Periplasmic Binding Proteins
and Disulfide Bonding Pattern
Z. Galen Wo and Robert E. Oswald
Department of Pharmacology
College of Veterinary Medicine
Cornell University
Ithaca, NY 14853 USA
Abstract
Low molecular weight kainate receptors from nonmammalian vertebrate brain belong to the ionotropic glutamate receptor superfamily. In this study, two previously cloned goldfish kainate receptor subunits (GFKARa and GFKARb) were transiently expressed in HEK 293 cells and their ligand binding properties and some underlying structural features were characterized. Both subunits form homomeric receptors which have high affinity for [3H]kainate (KD = 16 ± 3 nM and 31 ± 3 nM, respectively). Two conserved regions of the N-terminal extracellular domain (7VTTILE and 50DGKYG motifs) were found to be important determinants of L-glutamate binding. Glu12 is likely to interact directly with the ligand while at the same time interacting with Lys52. Mutation of Tyr53 dramatically affected ligand binding, and this residue may interact directly with agonist. Mutation of the three extracellular Cys residues of GFKARb indicated that the two conserved Cys residues (Cys305 and Cys385), located between two transmembrane segments, form a solvent accessible disulfide bond. A deletion mutant lacking the originally proposed "second transmembrane domain" was efficiently expressed and retains the overall ligand binding properties of wild type GFKARa. These data lend further evidence in support of a model of glutamate receptor topology with three transmembrane segments and reveal several important structural features of the extracellular ligand binding domain of the kainate receptors. These structural features are likely shared by other members of the iGluR superfamily.
Title Page
Introduction
Experimental Procedures
Results: L-Glutamate Binding Site
Results: Disulfide Bond
Results: Deletion of TMII
Discussion
References
Acknowledgements