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.

Two regions in the N-terminal domain of ionotropic glutamate receptor 3 form the subunit oligomerization interfaces that control subtype-specific receptor assembly.

The N-terminal domain (NTD) of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1-N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor delta2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.

The expression of dominant-negative subunits selectively suppresses neuronal AMPA and kainate receptors.

Glutamate-gated ion channels are widely expressed in neurons where they serve a host of cellular functions. An appealing, but yet unexplored, way to delineate the functions of particular glutamate receptor subtypes is to direct the expression of dominant-negative and gain-of-function mutant subunits. We tested the ability of two dominant-negative subunits, an alpha-amino-3-hydroxy-5-methyl-isoxazolproprionic acid receptor subunit and a kainate receptor subunit, to silence recombinant and neuronal glutamate receptors. Co-expression studies in non-neuronal cells indicated that the inclusion of a single mutant subunit was sufficient to silence the receptor. When expressed in cerebellar granule cells, the dominant-negative subunits silenced native channels in a subtype-specific fashion. Immunocytochemical staining of control and transfected neurons, as well as studies with a gain-of-function glutamate receptor-1 mutant, indicated that the mutant subunits were expressed at levels roughly equal to the total abundance of related native subunits, and both dominant-negatives suppressed native channel expression 60-65% when tested 24 h post-transfection. If co-assembly of the mutant subunits with related native subunits is combinatorial, this level of suppression gives receptor half-lives of approximately 20 h.

Palmitoylation of the GluR6 kainate receptor.

The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.