Dataset Information

Domain organization and function in GluK2 subtype kainate receptors.

ABSTRACT: Glutamate receptor ion channels (iGluRs) are excitatory neurotransmitter receptors with a unique molecular architecture in which the extracellular domains assemble as a dimer of dimers. The structure of individual dimer assemblies has been established previously for both the isolated ligand-binding domain (LBD) and more recently for the larger amino terminal domain (ATD). How these dimers pack to form tetrameric assemblies in intact iGluRs has remained controversial. Using recently solved crystal structures for the GluK2 kainate receptor ATD as a guide, we performed cysteine mutant cross-linking experiments in full-length tetrameric GluK2 to establish how the ATD packs in a dimer of dimers assembly. A similar approach, using a full-length AMPA receptor GluA2 crystal structure as a guide, was used to design cysteine mutant cross-links for the GluK2 LBD dimer of dimers assembly. The formation of cross-linked tetramers in full-length GluK2 by combinations of ATD and LBD mutants which individually produce only cross-linked dimers suggests that subunits in the ATD and LBD layers swap dimer partners. Functional studies reveal that cross-linking either the ATD or the LBD inhibits activation of GluK2 and that, in the LBD, cross-links within and between dimers have different effects. These results establish that kainate and AMPA receptors have a conserved extracellular architecture and provide insight into the role of individual dimer assemblies in activation of ion channel gating.

SUBMITTER: Das U

PROVIDER: S-EPMC2889583 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Domain organization and function in GluK2 subtype kainate receptors.

Das Utpal U Kumar Janesh J Mayer Mark L ML Plested Andrew J R AJ

Proceedings of the National Academy of Sciences of the United States of America 20100419 18

Glutamate receptor ion channels (iGluRs) are excitatory neurotransmitter receptors with a unique molecular architecture in which the extracellular domains assemble as a dimer of dimers. The structure of individual dimer assemblies has been established previously for both the isolated ligand-binding domain (LBD) and more recently for the larger amino terminal domain (ATD). How these dimers pack to form tetrameric assemblies in intact iGluRs has remained controversial. Using recently solved crysta ...[more]

PMID: 20404149

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Project description:Kainate receptors are ligand-gated ion channels that have two major roles in the central nervous system: they mediate a postsynaptic component of excitatory neurotransmission at some glutamatergic synapses and modulate transmitter release at both excitatory and inhibitory synapses. Accumulating evidence implicates kainate receptors in a variety of neuropathologies, including epilepsy, psychiatric disorders, developmental delay, and cognitive impairment. Here, to gain a deeper understanding of the conformational changes associated with agonist binding and channel opening, we generate a series of Cys substitutions in the GluK2 kainate receptor subunit, focusing on the M3 helices that line the ion pore and form the bundle-crossing gate at the extracellular mouth of the channel. Exposure to 50 µM Cd produces direct activation of homomeric mutant channels bearing Cys substitutions in (A657C), or adjacent to (L659C), the conserved SYTANLAAF motif. Activation by Cd is occluded by modification with 2-aminoethyl MTS (MTSEA), indicating that Cd binds directly and specifically to the substituted cysteines. Cd potency for the A657C mutation (EC50 = 10 µM) suggests that binding involves at least two coordinating residues, whereas weaker Cd potency for L659C (EC50 = 2 mM) implies that activation does not require tight coordination by multiple side chains for this substitution. Experiments with heteromeric and chimeric channels indicate that activation by Cd requires Cys substitution at only two of the four subunits within a tetrameric receptor and that activation is similar for substitution within subunits in either the A/C or B/D conformations. We develop simple kinetic models for the A657C substitution that reproduce several features of Cd activation as well as the low-affinity inhibition observed at higher Cd concentrations (5-20 mM). Together, these results demonstrate rapid and reversible channel activation, independent of agonist site occupancy, upon Cd binding to Cys side chains at two specific locations along the GluK2 inner helix.

Dataset Information

Domain organization and function in GluK2 subtype kainate receptors.

Publications

Domain organization and function in GluK2 subtype kainate receptors.

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