Calcium Permeability Of AMPA Receptors Research Articles

Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity.

Prion protein (PrP) mutations are linked to genetic prion diseases, a class of phenotypically heterogeneous neurodegenerative disorders with invariably fatal outcome. How mutant PrP triggers neurodegeneration is not known. Synaptic dysfunction precedes neuronal loss but it is not clear whether, and through which mechanisms, disruption of synaptic activity ultimately leads to neuronal death. Here we show that mutant PrP impairs the secretory trafficking of AMPA receptors (AMPARs). Specifically, intracellular retention of the GluA2 subunit results in synaptic exposure of GluA2-lacking, calcium-permeable AMPARs, leading to increased calcium permeability and enhanced sensitivity to excitotoxic cell death. Mutant PrPs linked to different genetic prion diseases affect AMPAR trafficking and function in different ways. Our findings identify AMPARs as pathogenic targets in genetic prion diseases, and support the involvement of excitotoxicity in neurodegeneration. They also suggest a mechanistic explanation for how different mutant PrPs may cause distinct disease phenotypes.

The Quaternary Structure of a Calcium-Permeable AMPA Receptor: Conservation of Shape and Symmetry across Functionally Distinct Subunit Assemblies

AMPA receptors (AMPA-Rs) are formed as heterotetrameric combinations of subunits known as GluR1–GluR4. The calcium permeability of AMPA-Rs is controlled by the identity of the amino-acid side chain contributed by each subunit at a key position in the conductance pathway, which can be either a glutamine (Q) or an arginine (R). Tetramers assembled only from Q-containing subunits are calcium permeable. In contrast, tetramers that incorporate R-containing subunits are calcium impermeable. Both forms play key roles in physiological and pathophysiological processes in the central nervous system. Here, using electron microscopy, we present the first quaternary structure of a calcium-permeable Q-homomeric AMPA-R. The receptor is elongated, with overall 2-fold symmetry and a large central vestibule. It is thus similar to the structure previously reported for an AMPA-R assembled exclusively from R-subunits. Both structures differ from those reported for brain-derived but urea-washed “native” AMPA-Rs, which exhibited multiple asymmetrical conformations. However, even transient exposure of our Q-homomeric AMPA-Rs to urea significantly attenuates the binding of a conformationally specific antibody. As a result, we propose a model in which all AMPA-Rs share a 2-fold symmetrical structure and in which subunit-dependent differences in assembly, trafficking, and electrophysiology are mediated within the framework of fundamentally similar quaternary conformations.

Auditory nerve input is not an absolute requirement for the expression, distribution and calcium permeability of AMPA receptors in the adult rat ventral cochlear nucleus

In order to understand whether glutamatergic excitatory presynaptic input is an absolute requirement for the adult regulation of postsynaptic glutamate receptors we analyzed if a period of 11 days of excitatory deprivation affects the expression, distribution and Ca 2+ permeability of AMPA receptor subunits in the ventral cochlear nucleus of the rat. Bilateral cochlear ablations were performed in 30-day-old rats. After 11 days of survival, immunohistochemistry for GluR1, GluR2/3 and GluR4 AMPA receptor subunits showed no changes in the normal pattern of distribution, with GluR2/3 and GluR4 immunoreactivity predominating, and little GluR1. No changes in the amount of these AMPA receptor subunits were found between normal and cochleotomized rats in Western blots. AMPA receptors lacking the GluR2 subunit are Ca 2+ permeable. Kainate-induced Co 2+ uptake histochemistry, which labels AMPA Ca 2+ permeable receptors, demonstrated no changes in somatic labeling intensity for Co 2+, 11 days after cochleotomy. Therefore, our data indicate that excitatory input is not an absolute requirement to maintain AMPA receptor subunit expression, distribution and functional properties such as Ca 2+ permeability in VCN neurons. Nevertheless, subtle changes in AMPA receptors through regulatory post-transductional mechanisms cannot be ruled out.

Bi-directional control of motor neuron dendrite remodeling by the calcium permeability of AMPA receptors

Motor neurons express particularly high levels of the AMPA receptor subunit GluR1(Q)flip (GluR1(Q)i) during the period in early postnatal life when their dendritic tree grows and becomes more branched. To investigate how GluR1-containing AMPA receptors contribute to dendrite morphogenesis, we characterized a mutant form of GluR1 (containing a histidine in the Q/R editing site) with unique electrophysiological properties. Most notably, AMPA receptors assembled from GluR1(H)i display less calcium permeability than AMPA receptors assembled from GluR1(Q)i. Expression of GluR1(Q)i in vivo or in vitro led to an increase in dendrite branching with no net change in the overall tree size while GluR1(H)i led to a loss of branches and a net reduction in overall tree size. GluR1(H)i-dependent dendrite atrophy is mediated by protein phosphatase 2B. The results suggest that the electrophysiological properties of cell surface AMPA receptors, specifically their permeability to calcium, can be a central determinant of whether the dendrites undergo activity-dependent branching or atrophy.

Tuning of RNA editing by ADAR is required in Drosophila

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel.

Calcium-Permeable AMPA Receptor Plasticity Is Mediated by Subunit-Specific Interactions with PICK1 and NSF

A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.

Aberrant Formation of Glutamate Receptor Complexes in Hippocampal Neurons of Mice Lacking the GluR2 AMPA Receptor Subunit

The number and type of receptors present at the postsynaptic membrane determine the response to the neurotransmitter released from the presynaptic terminal. Because most neurons receive multiple and distinct synaptic inputs and contain several different subtypes of receptors stimulated by the same neurotransmitter, the assembly and trafficking of receptors in neurons is a complex process involving many levels of regulation. To investigate the mechanism that neurons use to regulate the assembly of receptor subunits, we studied a GluR2 knock-out mouse. GluR2 is a critical subunit that controls calcium permeability of AMPA receptors and is present in most native AMPA receptors. Our data indicate that in the absence of GluR2, aberrant receptor complexes composed of GluR1 and GluR3 are formed in the hippocampus, and that there is an increased number of homomeric GluR1 and GluR3 receptors. We also show that these homomeric and heteromeric receptors are less efficiently expressed at the synapse. Our results show that GluR2 plays a critical role in controlling the assembly of AMPA receptors, and that the assembly of subunits may reflect the affinity of one subunit for another or the stability of intermediates in the assembly process. Therefore, GluR1 may have a greater preference for GluR2 than it does for GluR3.

The open channel blocking drug, IEM-1460, reveals functionally distinct α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors in rat brain neurons

The properties of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors were examined in various cell types isolated from young rat hippocampus, striatum and cerebellum using patch-clamp and fast application techniques. A dicationic adamantane derivative, IEM-1460, reversibly inhibited kainate-induced currents. In the presence of 100 μM IEM-1460, kainate currents in striatal giant cholinergic interneurons and hippocampal non-pyramidal neurons were inhibited by 95% and 81%, respectively, at V h=−70 mV. Striatal GABAergic principal cells, hippocampal pyramidal neurons and cerebellar Purkinje cells had low sensitivity to IEM-1460 (inhibition by 4–15%). Analysis of averaged data from the cell types studied revealed a highly significant positive correlation ( r=0.93, P<0.01) between percentage inhibition by 100 μM IEM-1460 and relative calcium permeability of AMPA receptors, P Ca/ P Na. Also, within each brain structure, the sensitivity of IEM-1460 block was lower the stronger the outward rectification of kainate currents. Some hippocampal neurons exhibited intermediate sensitivity to IEM-1460. Kainate currents were suppressed by 40% in the presence of 100 μM IEM-1460. Meanwhile, AMPA receptors in this cell type had low calcium permeability ( P Ca/ P Na=0.13) and demonstrated outwardly rectifying kainate currents. The interrelation of different properties of AMPA receptors considering their assembly is discussed. The data obtained suggest that IEM-1460 may be a convenient and promising marker of native AMPA receptor assembly: it selectively inhibits Ca 2+-permeable, GluR2-lacking AMPA receptors.

Correlation between the calcium permeability of ampa receptors and the expression of calcium-binding proteins

A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.

201 Correlation between the calcium permeability of AMPA receptors and the expression of calcium-binding proteins

The gap between on-road fuel efficiency and official fuel efficiency of passenger vehicles reported by manufacturers is widening, which would lead to the underestimate of real fuel consumption and biased projection of energy demand in the road transport sector. To investigate the gap between official and on-road fuel efficiency of passenger vehicles, we first built a comprehensive dataset of 393,108 valid observations, including vehicle system parameters, fuel-efficient technology, etc. Then we developed two regression models to quantify the driving factors of the increasing gap and slow improvement in on-road fuel efficiency, and the contribution of each factor to the gap. Results show that the fuel efficiency gap enlarged from 14.4% in 2010 to 25.1% in 2016, and the average on-road fuel efficiency did not have substantial improvement though the official fuel efficiency did. The fuel-efficient technologies have the greatest effect on both the fuel efficiency gap and on-road fuel efficiency among the factors considered in this study. Among the fuel-efficient technologies, hybrid electric vehicle would decrease the ratio of on-road to official fuel efficiency by 12.4% and improve the on-road fuel efficiency by 44.1%. Impacts of other fuel-efficient technologies were also assessed. Further government effort on policies to encourage fuel-efficient technologies for improving real on-road fuel efficiency is needed.

Synaptic Distribution of the AMPA-GluR2 Subunit and Its Colocalization with Calcium-Binding Proteins in Rat Cerebral Cortex: An Immunohistochemical Study Using a GluR2-Specific Monoclonal Antibody

Due to its role as the dominant AMPA receptor subunit in respect to regulation of calcium permeability, information on the neuronal localization of GluR2 is of particular importance, yet has been hampered by the lack of a GluR2-specific antibody. Monoclonal antibodies were raised against the putative N-terminal portion (amino acids 175–430) of GluR2, using the fusion protein linked to trpE as an antigen. Western blot analysis and immunocytochemistry of transiently transfected human embryonic kidney 293 cells unambiguously confirmed the specificity of monoclonal antibody 6C4 for GluR2, which did not recognize or cross-react with any other AMPA/Kainate GluR subunits expressed. 6C4 was used in immunohistochemical studies to characterize the regional, cellular, and subcellular distribution of the GluR2 subunit at the light and electron microscopic levels in rat hippocampus and somatosensory cortex and in colocalization studies with the three calcium-binding proteins: parvalbumin, calbindin, and calretinin. GluR2 was widely distributed in both pyramidal cells and interneurons. Asymmetric synapses were labeled on both spines and small dendritic shafts. In contrast to previous reports, our double labeling studies using monoclonal antibody 6C4 with polyclonal antisera against calcium-binding proteins demonstrated that 84–97% of parvalbumin and calbindin-immunoreactive and 45–66% of the calretinin-immunoreactive interneurons in CA1 and somatosensory cortex also contain GluR2. These data have important implications regarding heterogeneity in calcium permeability of AMPA receptors across cell types in neocortex and hippocampus, as well as for differential vulnerability to excitotoxic injury.