Received for publication May 16, 2008.
Revised June 26, 2008.
Accepted for publication July 28, 2008.

Mutations to the kainate receptor subunit GluR6 binding pocket that selectively affect domoate binding

Abstract

Kainate receptor responses to domoate are characterized by large steady-state currents and slow deactivation kinetics. To improve our understanding of these responses we mutated residues at the mouth of the agonist binding pocket of GluR6, using whole-cell electrophysiology to characterize the effects of the mutants. We identified two residues where mutations had significant ligand-specific effects. One, M691, forms a hydrogen bond that appears to facilitate domoate binding by affecting the main-chain conformation. We found that mutation of M691 to alanine significantly attenuated responses to domoate, while having no effect on responses to glutamate, confirming the importance of this main-chain interaction in GluR6. The second residue, V685, is located at the mouth of the binding pocket, adjacent to the domoate side-arm. Mutation of V685 to glutamine increased the rate of decay from steady-state responses to domoate by over 50-fold, while having no effect on the rate or extent of desensitization, or on the kinetics of responses to either glutamate or kainate. The V685Q mutant also significantly reduced the potencies of both glutamate (peak) and domoate (peak and steady-state). Empirical analysis using a basic kinetic model indicated that the V685Q phenotype could be fully explained by faster ligand dissociation. The V685Q mutant accelerated receptor deactivation without affecting either desensitization or gating, making it a potentially useful tool for further dissection of ligand binding and gating in kainate receptors.

Key words: Glutamate, Func. analysis receptor/ion channel mutants