Mutations to the kainate receptor subunit GluR6 binding pocket that selectively affect domoate binding.
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, Met691, forms a hydrogen bond that seems to facilitate domoate binding by affecting the main-chain conformation. We found that mutation of Met691 to alanine significantly attenuated responses to domoate but had no effect on responses to glutamate, confirming the importance of this main-chain interaction in GluR6. The second residue, Val685, is located at the mouth of the binding pocket, adjacent to the domoate side-arm. Mutation of Val685 to glutamine increased the rate of decay from steady-state responses to domoate by more than 50-fold but had 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.
Zhang
Yihong
Y
Department of Pharmacology, School of Biomedical Sciences, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK.
Nayeem
Naushaba
N
Green
Tim
T
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D018091:Receptors, AMPA / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D018092:Kainic Acid Receptors / Q000819:agonists / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D000097806:GluK2 Kainate Receptor
D000409:Alanine; D018698:Glutamic Acid; D005973:Glutamine; D007608:Kainic Acid; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D011994:Recombinant Proteins; D000097806:GluK2 Kainate Receptor; C012301:domoic acid