AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.
A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABA A receptors that contain the ␣5 subunit (␣5GABA A Rs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by ␣5GABA A Rs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not ␣5 null mutant (␣5Ϫ/Ϫ) mice. In addition, etomidate impaired memory performance of WT but not ␣5Ϫ/Ϫ mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The ␣5Ϫ/Ϫ mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and ␣5Ϫ/Ϫ mice. Deletion of the ␣5 subunit of the GABA A Rs reduced the amnestic but not the sedativehypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABA A R subtype pharmacology.
Ligand-gated ion channels gated by glutamate constitute the major excitatory neurotransmitter system in the mammalian brain. The functional modulation of GluR6, a kainate-activated glutamate receptor, by adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) was examined with receptors expressed in human embryonic kidney cells. Kainate-evoked currents underwent a rapid desensitization that was blocked by lectins. Kainate currents were potentiated by intracellular perfusion of PKA, and this potentiation was blocked by co-application of an inhibitory peptide. Site-directed mutagenesis was used to identify the site or sites of phosphorylation on GluR6. Although mutagenesis of two serine residues, Ser684 and Ser666, was required for complete abolition of the PKA-induced potentiation, Ser684 may be the preferred site of phosphorylation in native GluR6 receptor complexes. These results indicate that glutamate receptor function can be directly modulated by protein phosphorylation and suggest that a dynamic regulation of excitatory receptors could be associated with some forms of learning and memory in the mammalian brain.
A point mutation of the GluR␦2 (A654T) glutamate receptor subunit converts it into a functional channel, and a spontaneous mutation at this site is thought to be responsible for the neurodegeneration of neurons in the Lurcher mouse. This mutation is located in a hydrophobic region of the M3 domain of this subunit, and this alanine is conserved throughout many of the glutamate receptors. We show here that site-directed mutagenesis of the homologous alanine (A636T; GluR1-L c ) in the GluR1 AMPA receptor subunit alters its channel properties. The apparent potencies of both kainate and glutamate were increased 85-and 2000-fold, respectively. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)was converted from a competitive antagonist into a potent agonist. Our results demonstrate that a single amino acid within or near the putative second transmembrane region of the GluR1 subunit is critical for the binding/gating properties of this AMPA receptor.Ionotropic postsynaptic glutamate receptors are responsible for most of the rapid excitatory synaptic transmission in the central nervous system. The binding of glutamate to ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) 1 and Nmethyl-D-aspartic acid (NMDA) receptors generates excitatory synaptic currents in which the kinetics are determined by the relatively rapid gating of AMPA and the much slower gating of NMDA channels. Native channels consist of a heteromeric receptor composed of four subunits (1). A family of four different subunits (GluR1, -2, -3, and -4) can contribute to the formation of AMPA receptors, and each subunit can form functional homomeric channels (2, 3).A related subunit, GluR␦2, with 25% homology to AMPA receptors, is found in cerebellar Purkinje cells; a spontaneous mutation of the GluR␦2 subunit is responsible for the neurodegenerative phenotype of the Lurcher mouse (4, 5). The GluR␦2 subunit cannot form functional heteromeric channels (6, 7), but spontaneously gated currents can be recorded in the absence of an agonist for the Lurcher mutation (GluR␦2-L c ) (5). In this mutation, a substitution of a non-polar alanine with polar threonine occurs at position 654. This alanine is also conserved across a wide variety of glutamate receptors including the AMPA receptor subunits. It was postulated that neurons are lost in Lurcher mice as a consequence of spontaneous gating of GluR␦2-L c channels and the resulting chronic depolarization (5).The GluR␦2-L c mutation is of particular interest because it demonstrates that an apparently nonfunctional channel can be converted to a functional one simply by a point mutation in or near the putative second transmembrane region. Mutational analysis of AMPA receptor gating has been targeted primarily to regions of the N terminus as well as the extracellular loop (8), but little attention has been paid to the possible role of the second transmembrane region in the gating of AMPA channels. To explore the possible role of this region in channel gating, we constructed a mutated GluR1 (flop) subunit wit...
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