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A nondesensitizing kainate receptor point mutant.

ABSTRACT: Ionotropic glutamate receptor (iGluR) desensitization can be modulated by mutations that change the stability of a dimer formed by the agonist binding domain. Desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be blocked by a single point mutation (e.g., GluR2 L483Y) that stabilizes this dimer in an active conformation. In contrast, desensitization of kainate receptors can be slowed, but not blocked, by similar dimer interface mutations. Only covalent cross-linking via introduced disulfides has been previously shown to block kainate receptor desensitization completely. We have now identified an apparently nondesensitizing GluR6 point mutant (D776K) located at the apex of the ligand binding (S1S2) domain dimer interface. Asp776 is one of a cluster of four charged residues in this region that together mediate direct dimer interactions and contribute to the binding sites for one chloride and two sodium ions. Despite the localized +4 change in the net charge of the S1S2 dimer, the D776K mutation actually increased the thermodynamic stability of the dimer. Unlike GluR6 wild type, the D776K mutant is insensitive to external cations but retains sensitivity to external anions. We therefore hypothesize that the unexpected phenotype of this charge reversal mutation results from the substitution of the sodium ions bound within the dimer interface by the introduced lysine NH(3)(+) groups. The nondesensitizing D776K mutant provides insights into kainate receptor gating and represents a potentially useful new tool for dissecting kainate receptor function.

SUBMITTER: Nayeem N

PROVIDER: S-EPMC2730386 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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A nondesensitizing kainate receptor point mutant.

Nayeem Naushaba N Zhang Yihong Y Schweppe Devin K DK Madden Dean R DR Green Tim T

Molecular pharmacology 20090626 3

Ionotropic glutamate receptor (iGluR) desensitization can be modulated by mutations that change the stability of a dimer formed by the agonist binding domain. Desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be blocked by a single point mutation (e.g., GluR2 L483Y) that stabilizes this dimer in an active conformation. In contrast, desensitization of kainate receptors can be slowed, but not blocked, by similar dimer interface mutations. Only covalent cross ...[more]

PMID: 19561126

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Project description:GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the disease (autism) mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether the M867I mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 kainate receptor (rGluK2) as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ~1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminal sequence. By comparing the kinetic data of the wild-type human and rat GluK2 receptors, we also find that the human GluK2 has a ~3-fold smaller channel-opening rate constant but an identical channel-closing rate constant and thus a channel-opening probability of 0.85 vs 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K(1) for hGluK2, like the EC(50) value, is ~2-fold lower than rGluK2. Our results therefore suggest that the human GluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms share 99% sequence homology.

Dataset Information

A nondesensitizing kainate receptor point mutant.

Publications

A nondesensitizing kainate receptor point mutant.

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