Altered activity-rest patterns in mice with a human autosomal-dominant nocturnal frontal lobe epilepsy mutation in the ß2 nicotinic receptor.

High-affinity nicotinic receptors containing ß2 subunits (ß2*) are widely expressed in the brain, modulating many neuronal processes and contributing to neuropathologies such as Alzheimer's disease, Parkinson's disease and epilepsy. Mutations in both the α4 and ß2 subunits are associated with a rare partial epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In this study, we introduced one such human missense mutation into the mouse genome to generate a knock-in strain carrying a valine-to-leucine mutation ß2V287L. ß2(V287L) mice were viable and born at an expected Mendelian ratio. Surprisingly, mice did not show an overt seizure phenotype; however, homozygous mice did show significant alterations in their activity-rest patterns. This was manifest as an increase in activity during the light cycle suggestive of disturbances in the normal sleep patterns of mice; a parallel phenotype to that found in human ADNFLE patients. Consistent with the role of nicotinic receptors in reward pathways, we found that ß2(V287L) mice did not develop a normal proclivity to voluntary wheel running, a model for natural reward. Anxiety-related behaviors were also affected by the V287L mutation. Mutant mice spent more time in the open arms on the elevated plus maze suggesting that they had reduced levels of anxiety. Together, these findings emphasize several important roles of ß2* nicotinic receptors in complex biological processes including the activity-rest cycle, natural reward and anxiety.

Identification of a site in glutamate receptor subunits that controls calcium permeability.

The neurotransmitter glutamate mediates excitatory synaptic transmission throughout the brain. A family of genes encoding subunits of the non-N-methyl-D-aspartate (non-NMDA) type of glutamate receptor has been cloned. Some combinations of these subunits assemble into receptors with a substantial permeability to calcium, whereas others do not. To investigate the structural features that control ion permeation through these ligand-gated channels, mutant receptor subunits with single-amino acid changes were constructed. Mutation of a certain amino acid that results in a net charge change (from glutamine to arginine or vice versa) alters both the current-voltage relation and the calcium permeability of non-NMDA receptors. A site has thus been identified that regulates the permeation properties of these glutamate receptors.

Control of proton sensitivity of the NMDA receptor by RNA splicing and polyamines.

The function of the N-methyl-D-aspartate (NMDA)-preferring glutamate receptor can be regulated by extracellular pH, a process that may be important during ischemia in the brain or during seizures. Protons inhibit NMDA receptor function by 50 percent at pH 7.3 through interactions with the NR1 subunit, and both polyamines and NR1 exon 5 potentiate receptor function through relief of the tonic proton inhibition present at physiological pH. A single amino acid (lysine 211) was identified that mediates the effects of exon 5 in the rat brain. Electroneutral substitutions at this position restored pH sensitivity and, consequently, polyamine relief of tonic inhibition. This effect, together with the structural similarities between polyamines and the surface loop encoded by exon 5, suggest that exon 5 may act as a tethered pH-sensitive constitutive modulator of NMDA receptor function.

Autoantibodies to glutamate receptor GluR3 in Rasmussen's encephalitis.

Rasmussen's encephalitis is a progressive childhood disease of unknown cause characterized by severe epilepsy, hemiplegia, dementia, and inflammation of the brain. During efforts to raise antibodies to recombinant glutamate receptors (GluRs), behaviors typical of seizures and histopathologic features mimicking Rasmussen's encephalitis were found in two rabbits immunized with GluR3 protein. A correlation was found between the presence of Rasmussen's encephalitis and serum antibodies to GluR3 detected by protein immunoblot analysis and by immunoreactivity to transfected cells expressing GluR3. Repeated plasma exchanges in one seriously ill child transiently reduced serum titers of GluR3 antibodies, decreased seizure frequency, and improved neurologic function. Thus, GluR3 is an autoantigen in Rasmussen's encephalitis, and an autoimmune process may underlie this disease.

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.

Role of protein kinase C phosphorylation in rapid desensitization of metabotropic glutamate receptor 5.

Metabotropic glutamate receptors (mGluRs) coupled to phosphoinositide hydrolysis desensitize in response to prolonged or repeated agonist exposure, and evidence suggests that this involves activation of protein kinase C (PKC). The present studies were undertaken to determine if cloned mGluR5 undergoes similar PKC-mediated desensitization and to investigate the molecular mechanism underlying PKC-induced desensitization. In Xenopus oocytes, both mGluR5a and mGluR5b showed pronounced desensitization in response to a brief activation by glutamate. Pharmacological studies clearly suggest that this desensitization requires PKC-mediated phosphorylation. Analysis of PKC consensus phosphorylation site mutants suggests that PKC phosphorylates mGluR5 at multiple sites to induce a relatively rapid form of desensitization. Because mGluRs play important roles in synaptic plasticity and in excitotoxicity, this desensitization may be involved in the dynamic regulation of these processes.

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.

N-terminal domains in the NR2 subunit control desensitization of NMDA receptors.

Recent molecular studies of glutamate channels have provided increasingly detailed models of the agonist-binding site and of the channel pore. However, little information is available on the domains involved in channel gating. We examined the molecular determinants for the NR2-subunit specificity of glycine-independent desensitization of NMDA channels using NR2C/NR2A chimeric subunits expressed in HEK 293 cells. We show that glycine-independent desensitization is controlled by N-terminal domains of the NR2 subunit that flank the putative agonist-binding domain: a four amino acid (aa) segment immediately preceding the first transmembrane domain (M1) and a region containing the leucine/isoleucine/valine-binding protein-like (LIVBP-like) domain. Our results provide evidence for a functional role of the region containing the LIVBP-like domain in glutamate receptor channels. We suggest that the pre-M1 segment, presumably situated near the entrance to the pore, serves as a dynamic link between ligand binding and channel gating.

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.

Identification of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor.

Modulation of NMDA-mediated responses by oxidizing and reducing reagents has been described in a variety of neuronal preparations. Here, we report that NMDA-gated currents of oocytes expressing heteromeric NMDA receptors are also modulated by sulfhydryl redox reagents. Each cysteine residue in the NMDAR1 (NR1) subunit and each conserved NMDAR2 (NR2) cysteine residue in a prototypical subunit (NR2B) was tested for its role in redox modulation. We have identified 2 cysteines in the NR1 subunit that are required for redox modulation of NMDA-gated currents in oocytes expressing NR1-NR2B, NR1-NR2C, or NR1-NR2D receptors. Mutation of these same 2 cysteines also eliminated potentiation by spermine and shifted the IC50 for H+ inhibition and the EC50 for NMDA. Redox modulation of heteromeric NR1-NR2A receptors appeared to be different from that of the other heteromeric receptors, indicating the presence of one or more unique redox modulatory sites on NR1-NR2A receptors.

Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins.

By exchanging portions of the AMPA receptor subunit GluR3 and the kainate receptor subunit GluR6, we have identified two discontinuous segments of approximately 150 amino acid residues each that control the agonist pharmacology of these glutamate receptors. The first segment (S1) is adjacent and N-terminal to the putative transmembrane domain 1 (TM1), whereas the second segment (S2) is located between the putative TM3 and TM4. Only the simultaneous exchange of S1 and S2 converts the pharmacological profile of the recipient to that of the donor subunit. The two segments identified in this study share sequence similarities with the ligand-binding site of several bacterial periplasmic amino acid-binding proteins. Based on the X-ray structure of these proteins, we propose a model for the glutamate-binding site of ionotropic glutamate receptors.

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.

Inner ear defects induced by null mutation of the isk gene.

The isk gene is expressed in many tissues. Pharmacological evidence from the inner ear suggests that isk mediates potassium secretion into the endolymph. To examine the consequences of IsK null mutation on inner ear function, and to produce a system useful for examining the role(s) IsK plays elsewhere, we have produced a mouse strain that carries a disrupted isk locus. Knockout mice exhibit classic shaker/waltzer behavior. Hair cells degenerate, but those of different inner ear organs degenerate at different times. Functionally, we show that in mice lacking isk, the strial marginal cells and the vestibular dark cells of the inner ear are unable to generate an equivalent short circuit current in vitro, indicating a lack of transepithelial potassium secretion.

Role of alpha9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation.

Cochlear outer hair cells (OHCs) express alpha9 nACh receptors and are contacted by descending, predominately cholinergic, efferent fibers originating in the CNS. Mice carrying a null mutation for the nACh alpha9 gene were produced to investigate its role(s) in auditory processing and development of hair cell innervation. In alpha9 knockout mice, most OHCs were innervated by one large terminal instead of multiple smaller terminals as in wild types, suggesting a role for the nACh alpha9 subunit in development of mature synaptic connections. Alpha9 knockout mice also failed to show suppression of cochlear responses (compound action potentials, distortion product otoacoustic emissions) during efferent fiber activation, demonstrating the key role alpha9 receptors play in mediating the only known effects of the olivocochlear system.

The role of RNA editing of kainate receptors in synaptic plasticity and seizures.

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.

A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux.

Tyrosine phosphorylation can upregulate NMDA receptor activity during pathological and physiological alterations of synaptic strength. Here we describe downregulation of recombinant NR1/2A receptors by tyrosine dephosphorylation that requires agonist binding, but is independent of ion flux. The tyrosine residues involved in this new form of NMDA receptor modulation likely form a 'ring' adjacent to the last transmembrane domain. The downregulation was due to a reduction in the number of functional channels, and was blocked by co-expressing a dominant-negative mu2-subunit of the clathrin-adaptor protein AP-2. Our results provide a mechanism by which synaptic NMDA receptors can be modulated in a use-dependent manner even when the postsynaptic membrane is not sufficiently depolarized to relieve channel block by magnesium ions.

Activation of NMDA receptors reverses desensitization of mGluR5 in native and recombinant systems.

The metabotropic glutamate receptor, mGluR5, has a critical role in induction of NMDA-receptor-dependent forms of synaptic plasticity and excitotoxicity. This is likely mediated by a reciprocal positive-feedback interaction between these two glutamate receptor subtypes in which activation of mGluR5 potentiates NMDA receptor currents and NMDA receptor activation potentiates mGluR5-mediated responses. We have investigated the mechanism by which NMDA receptor activation modulates mGluR5 function and find evidence that this response is mediated by activation of a protein phosphatase and a resultant dephosphorylation of protein kinase C phosphorylation sites on mGluR5. This form of neuromodulation may be important in a number of normal and pathological processes that involve activation of the NMDA receptor.

NMDA-Dependent modulation of hippocampal kainate receptors by calcineurin and Ca(2+)/calmodulin-dependent protein kinase.

Neurotransmitter receptor function can be influenced by the phosphorylation state of the receptor or of associated proteins. Here we show that kainate receptors expressed in cultured hippocampal neurons can be modulated by Ca(2+)/calmodulin-dependent phosphatase (calcineurin) and Ca(2+)/calmodulin-dependent kinase (CaMK). Ca(2+) influx through NMDA receptor or voltage-sensitive calcium channels resulted in a transient depression of the kainate receptor current. This calcium-induced depression of the kainate receptor current depended on the activation of the phosphatase calcineurin. The amplitude of the kainate receptor currents returned to the baseline level in approximately 9 sec (tau = 3.6 sec), and the recovery of the current amplitude depended on CaMK activity. The effect on kainate receptor currents was dependent on the frequency of NMDA receptor activation. Although low-frequency (0.1 Hz) NMDA application induced depression followed by recovery of the kainate receptor currents, higher frequency (1 Hz) NMDA applications induced a more prolonged depression. Kainate receptors have been shown to modulate synaptic transmission by both presynaptic and postsynaptic mechanisms. Our results suggest that synaptic activity mediated by NMDA receptors, or other routes of Ca(2+) influx, may, in turn, modulate the function of kainate receptors.

Unequal expression of allelic kainate receptor GluR7 mRNAs in human brains.

We describe here the first example of an exonic polymorphism that affects the primary structure of a human ionotropic glutamate receptor. The human kainate receptor GluR7 gene contains a thymine (T)/guanine (G) nucleotide variation that determines a serine or alanine at position 310 in the extracellular region of GluR7 receptor subunits. Our finding contrasts with a previous report that suggested that GluR7 transcripts were RNA-edited at this site. Whole-cell patch-clamp recordings did not detect differences in receptor activation and desensitization between the human GluR7 receptor isoforms expressed in HEK-293 cells. Analysis of 41 tissue samples obtained from 30 human brains revealed expression level differences between GluR7 alleles expressed in the same brain. The expression level of the allelic GluR7 mRNAs differed in 27 samples from 1.2- to 12.7-fold. Unequal expression level of allelic mRNAs is characteristic for genes that are affected by genomic imprinting or that contain mutations. Genomic imprinting in most cases is conserved between human and mice. However, we did not detect unequal expression of allelic GluR7 mRNAs in mice. Our results are important for future studies that explore a potential role or roles for GluR7 receptors in the brain and for neurological disorders.

Identification of the kainate receptor subunits underlying modulation of excitatory synaptic transmission in the CA3 region of the hippocampus.

To understand the physiological role of kainate receptors and their participation in seizure induction in animal models of epilepsy, it will be necessary to develop a comprehensive description of their action in the CA3 region of the hippocampus. Activation of presynaptic kainate receptors depresses excitatory synaptic transmission at mossy fiber and associational-commissural inputs to CA3 pyramidal neurons (Vignes et al., 1998; Bortolotto et al., 1999; Kamiya and Ozawa, 2000). In this study, we use gene-targeted mice lacking glutamate receptor 5 (GluR5) or GluR6 kainate receptor subunits to identify the receptor subunits that comprise the kainate receptors responsible for presynaptic modulation of CA3 transmission. We found that bath application of kainate (3 microm) profoundly reduced EPSCs at mossy fiber and collateral synapses in neurons from wild-type and GluR5(-/-) mice but had no effect on EPSCs in neurons from GluR6(-/-) mice. These results therefore contrast with previous studies that supported a role for GluR5-containing receptors at mossy fiber and associational-commissural synapses (Vignes et al., 1998; Bortolotto et al., 1999). Surprisingly, at perforant path synapses kainate receptor activation enhanced transmission; this potentiation was abolished in both GluR5 and GluR6 knock-out mice. Kainate receptors thus play multiple and complex roles to modulate excitatory synaptic transmission in the CA3 region of the hippocampus.