Geoffrey T. Swanson

Intracellular spermine confers rectification on rat calcium‐permeable AMPA and kainate receptors.

1. Whole‐cell recordings were made from cerebellar granule cells cultured in high‐K+ medium to induce expression of Ca(2+)‐permeable AMPA receptors. Current‐voltage (I‐V) plots of agonist‐evoked responses showed varying degrees of inward rectification, but became linear within 5‐10 min. 2. Recombinant Ca(2+)‐permeable kainate receptors, composed of GluR6(Q)/KA‐2 subunits, exhibited rectifying whole‐cell I‐V plots that became linear in outside‐out patches. 3. Loss of rectification in granule cells was prevented by including 100 microM spermine in the pipette; the degree of rectification was then correlated with Ca2+ permeability. 4. Spermine also prevented loss of rectification in patches containing GluR6(Q)/KA‐2 receptors (IC50, 1.7 microM). 5. We suggest that spermine, or a similar cellular constituent, may act as a cytoplasmic factor conferring inward rectification on Ca(2+)‐permeable non‐NMDA receptors, and that ‘washout’ of this factor underlies the observed loss of rectification.

Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus.

Kainate receptors alter the excitability of mossy fiber axons and have been reported to play a role in the induction of long-term potentiation (LTP) at mossy fiber synapses in the hippocampus. These previous studies have relied primarily on the use of compounds whose selectivity is unclear. In this report, we investigate short- and long-term facilitation of mossy fiber synaptic transmission in kainate receptor knockout mice. We find that LTP is reduced in mice lacking the GluR6, but not the GluR5, kainate receptor subunit. Additionally, short-term synaptic facilitation is impaired in GluR6 knockout mice, suggesting that kainate receptors act as presynaptic autoreceptors on mossy fiber terminals to facilitate synaptic transmission. These data demonstrate that kainate receptors containing the GluR6 subunit are important modulators of mossy fiber synaptic strength.

Rat GluR7 and a Carboxy-Terminal Splice Variant, GluR7b, Are Functional Kainate Receptor Subunits with a Low Sensitivity to Glutamate

Glutamate receptors of the kainate-preferring subtype have recently been shown to mediate synaptic transmission in the hippocampus. The low-affinity kainate receptor subunit GluR7 was found to be nonfunctional in previous studies. We report here that the GluR7 subunit and a novel carboxy-terminal splice variant, GluR7b, are functional glutamate receptors with unique pharmacological properties. In particular, glutamate exhibits a 10-fold lower potency for (non-desensitized) GluR7-mediated currents as compared to other non-NMDA receptor channels. These data will facilitate understanding of the distinct role played by GluR7 receptors in synaptic transmission.

Subunit composition of kainate receptors in hippocampal interneurons

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.

Effect of RNA editing and subunit co‐assembly single‐channel properties of recombinant kainate receptors.

1. Patch‐clamp methods have been used to examine single‐channel properties of recombinant GluR5 and GluR6 kainate‐preferring glutamate receptors which differ in a single amino acid residue as a result of RNA editing at the Q/R (glutamine/arginine) site. Subunits were expressed alone or in combination with the high‐affinity kainate receptor subunit KA ‐ 2 in transfected human embryonic kidney (HEK‐293) cells. 2. In outside‐out patches, unedited homomeric GluR6(Q) receptors exhibited directly resolved domoate‐activated single‐channel conductances of 8, 15 and 25 pS. Variance analysis of GluR6(Q) responses gave a mean conductance of 5.4 pS, while the edited isoform GluR6(R) had an unusually low channel conductance (225 fS). 3. Homomeric channels composed of GluR5(Q) subunits exhibited three conductance states of 5, 9 and 14 pS characterized by prolonged burst activations in the presence of domoate. In contrast, the GluR5(R) subunit, which has not previously been reported to form functional homomeric receptors, had an extremely low conductance (< 200 fS). 4. Heteromeric GluR6(Q)/KA‐2 kainate receptors gave single‐channel events indistinguishible from homomeric GluR6(Q) channels. Conversely, openings produced by GluR5(Q)KA‐2 and GluR5(Q) receptors differed from each other in their kinetic properties. The primary effect of co‐expression of KA‐2 with GluR5(Q) was a dramatic shortening in channel burst length. 5. Spectral and variance analyses were used to estimate mean single‐channel conductances of heteromeric edited receptor‐channels; channel conductances were 950 fS for GluR5(R)KA‐2 receptors and 700 fS for GluR6(R)/KA‐2 receptors. Both receptor types had significantly higher conductances than the respective homomeric channels, GluR5(R) and GluR6(R). 6. We conclude that Q/R site editing dramatically reduces single‐channel conductance. Furthermore, we find similarity between the kainate receptor‐channels described in sensory neurones and the recombinant GluR5(Q) homomeric channel. Characterization of recombinant single‐channel properties could therefore aid identification of the native kainate receptors.

Kainate receptors coming of age: milestones of two decades of research

Two decades have passed since the first report of the cloning of a kainate-type glutamate receptor (KAR) subunit. The intervening years have seen a rapid growth in our understanding of the biophysical properties and function of KARs in the brain. This research has led to an appreciation that KARs play very distinct roles at synapses relative to other members of the glutamate-gated ion channel receptor family, despite structural and functional commonalities. The surprisingly diverse and complex nature of KAR signaling underlies their unique impact upon neuronal networks through their direct and indirect effects on synaptic transmission, and their prominent role in regulating cell excitability. This review pieces together highlights from the two decades of research subsequent to the cloning of the first subunit, and provides an overview of our current understanding of the role of KARs in the CNS and their potential importance to neurological and neuropsychiatric disorders.

Distribution of Kainate Receptor Subunits at Hippocampal Mossy Fiber Synapses

Kainate receptors function as mediators of postsynaptic currents and as presynaptic modulators of synaptic transmission at mossy fiber synapses. Despite intense research into the physiological properties of mossy fiber kainate receptors, their subunit composition in the presynaptic and postsynaptic compartments is unclear. Here we describe the distribution of kainate receptor subunits in mossy fiber synapses using subunit-selective antibodies and knock-out mice. We provide morphological evidence for the presynaptic localization of KA1 and KA2 receptor subunits at mossy fiber synapses. Immunogold staining for KA1 and KA2 was commonly seen at synaptic contacts and in vesicular structures. Postsynaptic labeling in dendritic spines was also observed. Although KA1 predominantly showed presynaptic localization, KA2 was concentrated to a greater degree on postsynaptic membranes. Both subunits coimmunoprecipitated from hippocampal membrane extracts with GluR6 but not GluR7 subunits. These results demonstrate that KA1 and KA2 subunits are localized presynaptically and postsynaptically at mossy fiber synapses where they most likely coassemble with GluR6 subunits to form functional heteromeric kainate receptor complexes.

Rapid enhancement of two-step wiring plasticity by estrogen and NMDA receptor activity

Cortical information storage requires combined changes in connectivity and synaptic strength between neurons, but the signaling mechanisms underlying this two-step wiring plasticity are unknown. Because acute 17β-estradiol (E2) modulates cortical memory, we examined its effects on spine morphogenesis, AMPA receptor trafficking, and GTPase signaling in cortical neurons. Acute E2 application resulted in a rapid, transient increase in spine density, accompanied by temporary formation of silent synapses through reduced surface GluR1. These rapid effects of E2 were dependent on a Rap/AF-6/ERK1/2 pathway. Intriguingly, NMDA receptor (NMDAR) activation after E2 treatment potentiated silent synapses and elevated spine density for as long as 24 h. Hence, we show that E2 transiently increases neuronal connectivity by inducing dynamic nascent spines that “sample” the surrounding neuropil and that subsequent NMDAR activity is sufficient to stabilize or “hold” E2-mediated effects. This work describes a form of two-step wiring plasticity relevant for cortical memory and identifies targets that may facilitate recovery from brain injuries.

Identification of Amino Acid Residues that Control Functional Behavior in GluR5 and GluR6 Kainate Receptors

GluR5 and GluR6 kainate receptors differ in their responses to a variety of agonists, despite their relatively high primary sequence homology. We carried out a structure-function study to identify amino acids underlying these divergent responses. Patch clamp analysis of chimeric GluR5-GluR6 receptors indicated that several functionally dominant sites were localized to the C-terminal side of M1. All nonconserved amino acids in the region between M3 and M4 of GluR6 were then individually mutated to their GluR5 counterparts. We found that a single amino acid (N721 in GluR6) controls both AMPA sensitivity and domoate deactivation rates. Additionally, mutation of A689 in GluR6 slowed kainate desensitization. These functional effects were accompanied by alterations in binding affinities. These results support a critical role for these residues in receptor binding and gating activity.

High-Affinity Kainate Receptor Subunits Are Necessary for Ionotropic but Not Metabotropic Signaling

Kainate receptors signal through both ionotropic and metabotropic pathways. The high-affinity subunits, GluK4 and GluK5, are unique among the five receptor subunits, as they do not form homomeric receptors but modify the properties of heteromeric assemblies. Disruption of the Grik4 gene locus resulted in a significant reduction in synaptic kainate receptor currents. Moreover, ablation of GluK4 and GluK5 caused complete loss of synaptic ionotropic kainate receptor function. The principal subunits were distributed away from postsynaptic densities and presynaptic active zones. There was also a profound alteration in the activation properties of the remaining kainate receptors. Despite this, kainate receptor-mediated inhibition of the slow afterhyperpolarization current (I(sAHP)), which is dependent on metabotropic pathways, was intact in GluK4/GluK5 knockout mice. These results uncover a previously unknown obligatory role for the high-affinity subunits for ionotropic kainate receptor function and further demonstrate that kainate receptor participation in metabotropic signaling pathways does not require their classic role as ion channels.