Activation of N-methyl-D-aspartate receptors reverses desensitization of metabotropic glutamate receptor, mGluR5, in native and recombinant systems.
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Animals↗
Biomedical subjects
S F Heinemann
Publications and source records attributed to S F Heinemann.
Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors.
Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and alpha-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of alpha-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of alpha-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or alpha-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from alpha-actinin by two distinct but converging calcium-dependent processes: competitive displacement of alpha-actinin by calmodulin and reduction in the affinity of alpha-actinin for C0 after binding of calcium to alpha-actinin.
Actinin↗
Lectin-induced inhibition of desensitization of the kainate receptor GluR6 depends on the activation state and can be mediated by a single native or ectopic N-linked carbohydrate side chain.
The ionotropic glutamate receptor GluR6 exhibits strongly and rapidly desensitizing current responses. Treatment of heterologically expressed GluR6 with the lectin concanavalin A (ConA) in Xenopus oocytes as well as in human embryonic kidney-293 cells results in a considerable increase of the steady-state current, presumably by inhibiting receptor desensitization. In the present study, we investigated the molecular basis of this effect using a systematic mutagenesis approach. We found that although N-glycosylation is an absolute prerequisite for the lectin-mediated inhibition of desensitization, no single one of the nine extracellular consensus sites for N-glycosylation of GluR6 is required. Rather, each of the nine N-linked carbohydrate side chains is independently capable of modulatory interaction with the lectin. Moreover, even artificially introduced N-glycosylation sites can substitute for native sites. Thus, the specific site of the lectin binding does not appear to be important for its desensitization-inhibiting action. Furthermore, we show that the extent of the receptor's ConA sensitivity depends on its state of activation, because the desensitized GluR6 exhibits significantly lower lectin sensitivity than the nondesensitized receptor. We conclude that binding of ConA "locks" the receptor in the activatable state, thereby inhibiting conformational changes required to shift the receptor to the desensitized state.
Animals↗
Kainate receptor-mediated responses in the CA1 field of wild-type and GluR6-deficient mice.
Kainate receptors are abundantly expressed in the hippocampus. Mice with disruption of kainate receptor subunits allow the genetic dissection of the role of each kainate receptor subunits in the synaptic physiology of the hippocampus, as well as in excitotoxic processes. We have compared the action of domoate and kainate on CA1 pyramidal neurons in slices from wild-type and GluR6-/- mice. The difference in the amplitude of inward currents evoked by domoate and kainate between wild-type and GluR6-/- mice demonstrates the presence of functional kainate receptors in CA1 pyramidal neurons. Block of domoate-activated inward currents by the AMPA receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo(F)quinoxaline (1 microM) and 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine) (GYKI 53655) (50 microM) is complete in GluR6-/- mice but only partial in wild-type mice. In the presence of GYKI 53655, kainate receptor activation dramatically increases the frequency of spontaneous IPSCs in CA1 pyramidal cells from wild-type, as well as GluR6-/-, mice. This results from the kainate receptor-mediated activation of a sustained inward current and an increased action potential firing in afferent GABAergic interneurons of the CA1 field. These effects are observed in wild-type, as well as GluR6-/-, mice. Kainate receptors also decrease the amplitude of evoked IPSCs in CA1 pyramidal cells by increasing synaptic failures in wild-type and GluR6-/- mice. These results indicate that in CA1 pyramidal cells, distinct subtypes of kainate receptors mediate several functionally antagonistic effects.
Action Potentials↗
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.
Animals↗
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.
Animals↗
Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells.
1. Kainate receptors with pharmacological properties similar to those of the GluR5 subunit have been shown to modulate inhibitory synaptic transmission in the CA1 region of the hippocampus. The kinetic properties of currents gated by GluR5 receptors have not been examined in detail. Here we describe several biophysical features of recombinant GluR5 receptors expressed in HEK293 cells. 2. We found that homomeric GluR5 receptors can exhibit striking inter-cell variability in channel kinetics in response to the agonists kainate and glutamate. Desensitization rates in response to kainate varied between individual cells by nearly 1000-fold (range, 1.5 ms to 1.5 s), while glutamate desensitization rates differed by 9-fold (range, 1.0 to 9.0 ms). 3. The time course of recovery from desensitization in response to glutamate also showed inter-cell variation. The majority of glutamate currents in GluR5-expressing cells recovered from desensitization with two widely separated exponential components: 50 +/- 10 ms and 5.1 +/- 1.0 s (contributing 37.6 % and 62.4 % of the sum of the exponential fits, respectively). In contrast, currents with the fastest desensitization kinetics had a recovery time course of 4.8 +/- 0.3 s. 4. Kainate receptors in murine dorsal root ganglion neurons are likely to be composed of homomeric GluR5 subunits. These receptor currents recovered from glutamate desensitization with a biexponential time course of 36 +/- 4 ms and 4.7 +/- 0.7 s. 5. These results suggest that aspects of GluR5 kainate receptor function are modulated by intracellular mechanism(s). At synapses such mechanisms could regulate the frequency- response relationship of synaptic kainate receptors by altering their rate of entry into and recovery from desensitization.
Animals↗
Direct effects of metabotropic glutamate receptor compounds on native and recombinant N-methyl-D-aspartate receptors.
The actions of glutamate in the central nervous system are mediated through interaction with fast activating ionotropic receptors and G protein-coupled metabotropic glutamate receptors (mGluRs). Studies of these receptors have relied on the availability of agonists and antagonists selective for each receptor class. Compounds that were thought to be selective for mGluRs have been extensively used to study the role of these receptors in the brain. Their use has implicated mGluRs in a wide range of physiological and pathological processes including the modulation of N-methyl-D-aspartate (NMDA) receptors and NMDA receptor-dependent processes. We report that some of the most commonly used mGluR compounds act as antagonists on NMDA receptors at concentrations commonly used to activate or block mGluRs. In addition, several of the drugs also act as agonists at higher concentrations due at least in part to high levels of contaminant amino acids. Our results indicate that caution should be used when using these drugs to study the roles of mGluRs in various NMDA-dependent processes. The antagonist effects were dependent on the concentration of the NMDA receptor coagonists, preventing reappraisal of previously published work.
Animals↗
Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice.
L-glutamate, the neurotransmitter of the majority of excitatory synapses in the brain, acts on three classes of ionotropic receptors: NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptors. Little is known about the physiological role of kainate receptors because in many experimental situations it is not possible to distinguish them from AMPA receptors. Mice with disrupted kainate receptor genes enable the study of the specific role of kainate receptors in synaptic transmission as well as in the neurotoxic effects of kainate. We have now generated mutant mice lacking the kainate-receptor subunit GluR6. The hippocampal neurons in the CA3 region of these mutant mice are much less sensitive to kainate. In addition, a postsynaptic kainate current evoked in CA3 neurons by a train of stimulation of the mossy fibre system is absent in the mutant. We find that GluR6-deficient mice are less susceptible to systemic administration of kainate, as judged by onset of seizures and by the activation of immediate early genes in the hippocampus. Our results indicate that kainate receptors containing the GluR6 subunit are important in synaptic transmission as well as in the epileptogenic effects of kainate.
Animals↗
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.
Animals↗
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.
Amino Acid Sequence↗
Molecular neurobiology and genetics: investigation of neural function and dysfunction.
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Animals↗
Effects of ethanol and anesthetics on type 1 and 5 metabotropic glutamate receptors expressed in Xenopus laevis oocytes.
Previous studies have demonstrated that ethanol and volatile anesthetics inhibit the function of some metabotropic (G protein-coupled) receptors, including the 5-hydroxytryptamine2 and muscarinic cholinergic receptors. The metabotropic glutamate receptors (mGluRs) show little sequence homology with most other metabotropic receptors and are important modulators of synaptic transmission in the mammalian central nervous system. It was of interest to determine drug actions on these receptors, and we investigated the effects of ethanol, halothane, the anesthetic compound F3 (1-chloro-1,2,2-trifluorocyclobutane), and the nonanesthetics F6 (1,2-dichlorohexafluorocyclobutane) and F8 (2,3-chlorooctafluorobutane) on the function of mGluR1 and mGluR5 expressed in Xenopus laevis oocytes. Halothane, F3, and ethanol inhibited mGluR5-induced Ca(2+)-dependent Cl- currents, yet pharmacologically relevant concentrations of these compounds had little effect on the glutamate-induced currents in the oocytes expressing mGluR1. F6 had inhibitory effects on both receptors, and F8 did not affect either mGluR1 or mGluR5 function. The protein kinase C (PKC) inhibitor GF109203X enhanced the glutamate-induced current, and the PKC activator phorbol-12-myristate-13-acetate inhibited this current in the oocytes expressing mGluR5, but these compounds had little effect on mGluR1 function. GF109203X abolished the inhibitory effects of halothane, F3, and ethanol on mGluR5s. Conversely, the phosphatase inhibitor calyculin A prolonged the action of halothane and ethanol. Furthermore, mutation of a PKC consensus site (Ser890) of mGluR5 abolished the inhibitory effects of halothane, F3, and ethanol. These results suggest that ethanol and volatile anesthetics inhibit mGluR5 because they promote PKC-mediated phosphorylation.
Anesthetics, Inhalation↗
Kainate receptors exhibit differential sensitivities to (S)-5-iodowillardiine.
Characterization of the role of kainate receptors in excitatory synaptic transmission has been hampered by a lack of subtype-selective pharmacological agents. (S)-5-Iodowillardiine (IW), an analog of willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione], a heterocyclic amino acid found in Acacia and Mimosa seeds, was previously shown to be highly potent on native kainate receptors in dorsal root ganglion neurons. We examined the responses evoked by IW from recombinant homomeric and heteromeric kainate receptors expressed in human embryonic kidney 293 cells. IW potently elicited currents from glutamate receptor 5 (GluR5)-expressing cells, but showed no activity on homomeric GluR6 or GluR7 receptors. Co-expression of these receptor subunits with KA-2 subunits produced receptors that were weakly sensitive to IW. GluR5/KA-2 receptors had a higher EC50 value than homomeric GluR5 and exhibited a much faster recovery from desensitization. Finally, we found that the IW selectivity for GluR5 compared with GluR6 was determined by amino acid 721, which was previously shown to control alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate sensitivity of these kainate receptor subunits. The pharmacological selectivity and commercial availability of IW suggests that this compound may be of use in characterizing the molecular constituents of native kainate receptor responses.
Alanine↗
Workshop on schizophrenia.
On November 29-30, 1995, the National Academy of Sciences and the Institute of Medicine brought together experts in schizophrenia and specialists in other areas of the biological sciences in a workshop aimed at promoting the application of the latest biological information to this clinical problem. The workshop paid particular attention to evidence of pathology in the brains of people with schizophrenia, and to the possibility that this reflects an abnormality in brain development that eventually leads to the appearance of symptoms. The participants were impressed with the complexity of the problem, and felt that multiple approaches would be required to understand this disease. They recommended that a major focus should be on the search for predisposing genes, but that there should be parallel research in many other areas.
Brain↗
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Animals↗