Cations but not anions regulate the responsiveness of kainate receptors.

Kainate-selective ionotropic glutamate receptors are unique among ligand-gated ion channels in their obligate requirement of external anions and cations for activation. Although it is established that the degree of kainate receptor (KAR) activation is shaped by the chemical nature of the agonist molecule, the possible complementary role of external ions has yet to be examined. Here we show that external cations but not anions regulate the responsiveness to a range of full and partial agonists acting on rat GluK2 receptors. This observation is unexpected as previous work has assumed anions and cations affect KARs in an identical manner through functionally coupled binding sites. However, our data demonstrate that anion- and cation-binding pockets behave discretely. We suggest cations uniquely regulate a pregating or flipping step that impacts the closed-cleft stability of the agonist-binding domain (ABD). This model departs from a previous proposal that KAR agonist efficacy is governed by the degree of closure elicited in the ABD by ligand binding. Our findings are, however, in line with recent studies on Cys-loop ligand-gated ion channels suggesting that the "flipping" mechanism has been conserved by structurally diverse ligand-gated ion channel families as a common means of regulating neurotransmitter behavior.

Functional characterization and in silico docking of full and partial GluK2 kainate receptor agonists.

Two structural models have been developed to explain how agonist binding leads to ionotropic glutamate receptor (iGluR) activation. At alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) iGluRs, full and partial agonists close the agonist-binding domain (ABD) to different degrees whereas agonist-induced domain closure is apparently fixed at N-methyl-D-aspartate receptors. Although kainate (KA) iGluRs are thought to behave like AMPA receptors, the issue has not been formally tested because of the paucity of available receptor agonists. Here we identify a series of structurally related full and partial agonists at GluK2 (formerly GluR6) KARs and predict their docking mode using the in silico ligand-docking program FITTED. As expected, the neurotransmitter L-Glu behaved as a full agonist but modest reduction (e.g., L-serine or L-aspartate) or elongation (e.g., L-alpha-aminoadipate) in chain length generated weak partial agonists. It is noteworthy that in silico ligand-docking predicted that most partial agonists select for the closed and not, as expected, the open or intermediate conformations of the GluK2 ABD. Experiments using concanavalin-A to directly report conformations in the intact GluK2 receptor support this prediction with the full agonist, L-Glu, indistinguishable in this regard from weak partial agonists, D- and L-Asp. Exceptions to this were KA and domoate, which failed to elicit full closure as a result of steric hindrance by a key tyrosine residue. Our data suggest that alternative structural models need to be considered to describe agonist behavior at KARs. Finally, our study identifies the responsiveness to several neurotransmitter candidates establishing the possibility that endogenous amino acids other than L-Glu may regulate native KARs at central synapses.

External ions are coactivators of kainate receptors.

The activation of ligand-gated ion channels is thought to depend solely on the binding of chemical neurotransmitters. In this study, we demonstrate that kainate (KA) ionotropic glutamate receptors (iGluRs) require not only the neurotransmitter L-glutamate (L-Glu) but also external sodium and chloride ions for activation. Removal of external ions traps KA receptors (KARs) in a novel inactive state that binds L-Glu with picomolar affinity. Moreover, occupancy of KARs by L-Glu precludes external ion binding, demonstrating crosstalk between ligand- and ion-binding sites. AMPA iGluRs function normally in the absence of external ions, revealing that even closely related iGluR subfamilies operate by distinct gating mechanisms. This behavior is interchangeable via a single amino acid residue that operates as a molecular switch to confer AMPA receptor behavior onto KARs. Our findings identify a novel allosteric site that singles out KARs from all other ligand-gated ion channels.

Concanavalin-A reports agonist-induced conformational changes in the intact GluR6 kainate receptor.

The agonist-binding domain of ionotropic glutamate receptors (iGluRs) has recently been crystallized as two polypeptide chains with a linker region. Although work on the structure of this isolated ligand-binding core has been invaluable, there is debate over how it relates to conformations adopted by intact receptors. iGluR crystals are proposed to represent the activated state as their degree of domain closure correlates well with agonist efficacy. However, iGluR crystals exhibit high agonist affinity that more closely matches that of desensitized receptors. Consequently, conformations adopted by iGluR crystals may represent this state. To test this, we have employed the plant lectin, concanavalin-A (Con-A) to report conformational changes elicited by kainate (KA) iGluR agonists during desensitization. When GluR6 KA receptors (KARs) were pre-incubated with Con-A, equilibrium responses evoked by the full agonist, l-glutamate (l-Glu), increased almost 30-fold. However, in the continued presence of l-Glu, Con-A exerted no effect suggesting that it has restricted access to its binding sites when the agonist is bound. However, Con-A does not discriminate well between agonist-bound or -unbound states with the weak partial agonist, domoate. Accessibility experiments with KA were intermediate in nature consistent with its equilibrium efficacy at GluR6 KARs. Our results suggest that full and partial agonists elicit distinct conformational changes in KARs during desensitization. This finding can be reconciled with crystallographic data if the agonist-binding domain adopts the same conformation in the activated and desensitized states. However, other interpretations are possible suggesting future work is required if this issue is to be resolved.