Abstract

Low-molecular-weight kainate receptors from nonmammalian vertebrate brain belong structurally to the ionotropic glutamate receptor superfamily. In this study, two previously cloned goldfish kainate receptor subunits (GFKAR alpha and GFKAR beta) were transiently expressed in human embryonic kidney 293 cells, and their ligand-binding properties and some associated structural features were characterized, resulting in the following findings. 1) Both subunits form homomeric receptors with high affinity for [3H]kainate (KD = 16 and 31 nM, respectively) and L-glutamate (KD = 2 and 40 microM, respectively). 2) A deletion mutant lacking the originally proposed second-transmembrane domain was efficiently expressed and retains the overall ligand-binding properties of wild-type GFKAR alpha, strongly indicating that this region is not a transmembrane domain. 3) Mutations of Q12, A53, and Y54 of GFKAR beta indicate that these three residues are important for ligand binding (particularly L-glutamate), which is consistent with the sequence homology to bacterial periplasmic binding proteins. 4) Mutation of the three extracellular cysteine residues of GFKAR beta indicated that the two conserved cysteine residues (C305 and C385), located between two transmembrane segments, form a solvent-accessible disulfide bond. Analysis of [3H]kainate binding to wild-type and cysteine mutations of GFKAR beta indicate that in the absence of the disulfide bond, the affinity for kainate is increased 3-fold. These data lend further evidence in support of a model of glutamate receptor topology with three transmembrane segments and reveal several general structural features of the extracellular ligand-binding domain of the kainate receptors. These results are consistent with the notion that the ligand-binding domain has close structural similarities to bacterial periplasmic binding proteins.