The GluK3 Ligand Binding Domain has a Zinc Binding Site at the Dimer Interface

Abstract

GluK3 receptors belong to the family of kainate subtype ligand-gated glutamate receptor ion channels. Experiments in Bordeaux revealed that zinc selectively potentiated glutamate evoked currents for GluK3 while responses for GluK2 were inhibited. Mutagenesis and analysis of chimeric GluK2/GluK3 receptors mapped the zinc-binding site to the S2 segment of the ligand-binding domain (LBD) in a region expected to form the interface between two GluK3 subunits in an LBD dimer assembly. Multiple sequence alignments, coupled with site directed mutagenesis, revealed that a GluK3 specific aspartate residue, D759, which is exchanged for glycine in GluK1, GluK2 and in AMPA receptors, was essential for zinc potentiation. To identify additional residues contributing to the zinc binding site we attempted to solve a GluK3 LBD dimer assembly crystal structure. This was hampered by formation of zinc mediated intermolecular contacts between LBD monomers that favored other, non-biologically relevant head to tail dimer assemblies in three crystal forms solved for GluK3 glutamate and kainate complexes. However, from these structures we could generate a model dimer assembly by least squares superimposition of two copies of a GluK3 monomer on a previously solved GluK2 LBD dimer structure. This revealed that adjustment of rotamers for D730, D759 and H762 would allow formation of intersubunit contacts with appropriate bonding distances for zinc coordination. Mutagenesis experiments confirm that D730 on one subunit, together with D759 and H762 in the dimer partner, together form the binding site for zinc potentiation. The model also revealed that zinc acts as a countercharge that neutralizes the negative charges of the two coordinating aspartate residues, thereby stabilizing the dimer assembly and reducing desensitization. These results suggest that zinc may act as an endogenous positive allosteric modulator of native GluK3 containing kainate receptors.