Activation and desensitization of ionotropic glutamate receptors by selectively triggering pre-existing motions.
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ABSTRACT:
SUBMITTER: Krieger J
PROVIDER: S-EPMC6107436 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPAR-like. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process.Rapid communication between cells in the nervous system depends on ion channels that are directly activated by neurotransmitter molecules. Here, we studied ionotropic glutamate receptors (iGluRs), which are ion channels activated by the neurotransmitter glutamate. By comparing the sequences of a vast number of iGluR proteins from diverse animal species, assisted by available structural information, we identified a highly conserved motif. We showed that a single amino acid difference in this motif between mammalian iGluR subtypes has dramatic effects on receptor function. These results have implications in both the evolution of synaptic function, as well as the role of iGluRs in health and disease.
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Project description:Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels essential to all aspects of brain function, including higher order processes such as learning and memory. For decades, electrophysiology was the primary means for characterizing the function of iGluRs and gaining mechanistic insight. Since the turn of the century, structures of isolated water-soluble domains and transmembrane-domain-containing constructs have provided the basis for formulating mechanistic hypotheses. Because these structures only represent sparse, often incomplete snapshots during iGluR activation, significant gaps in knowledge remain regarding structures, energetics, and dynamics of key substates along the functional processes. Some of these gaps have recently been filled by molecular dynamics simulations and theoretical modeling. In this Account, I describe our work in the latter arena toward characterizing iGluR gating motions and developing a formalism for calculating thermodynamic and kinetic properties of stationary gating. The structures of iGluR subunits have a highly modular architecture, in which the ligand-binding domain and the transmembrane domain are well separated and connected by flexible linkers. The ligand-binding domain in turn is composed of two subdomains. During activation, agonist binding induces the closure of the intersubdomain cleft. The cleft closure leads to the outward pulling of a linker tethered to the extracellular terminus of the major pore-lining helix of the transmembrane domain, thereby opening the channel. This activation model based on molecular dynamics simulations was validated by residue-specific information from electrophysiological data on cysteine mutants. A further critical test was made through introducing glycine insertions in the linker. Molecular dynamics simulations showed that, with lengthening by glycine insertions, the linker became less effective in pulling the pore-lining helix, leading to weaker stabilization of the channel-open state. In full agreement, single-channel recordings showed that the channel open probability decreased progressively as the linker was lengthened by glycine insertions. Crystal structures of ligand-binding domains showing different degrees of cleft closure between full and partial agonists suggested a simple mechanism for one subtype of iGluRs, but mysteries surrounded a second subtype, where the ligand-binding domains open to similar degrees when bound with either full or partial agonists. Our free energy simulations now suggest that broadening of the free energy basin for cleft closure is a plausible solution. A theoretical basis for these mechanistic hypotheses on partial agonisms was provided by a model for the free energy surface of a full receptor, where the stabilization by cleft closure is transmitted via the linker to the channel-open state. This model can be implemented by molecular dynamics simulations to predict thermodynamic and kinetics properties of stationary gating that are amenable to direct test by single-channel recordings. Close integration between computation and electrophysiology holds great promises in revealing the conformations of key substates in functional processes and the mechanisms of disease-associated mutations.
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Project description:The neurotransmitter glutamate mediates excitatory synaptic transmission by activating ionotropic glutamate receptors (iGluRs). In Caenorhabditis elegans, the GLR-1 receptor subunit is required for glutamate-gated current in a subset of interneurons that control avoidance behaviors. Current mediated by GLR-1-containing iGluRs depends on SOL-1, a transmembrane CUB-domain protein that immunoprecipitates with GLR-1. We have found that reconstitution of glutamate-gated current in heterologous cells depends on three proteins, STG-1 (a C. elegans stargazin-like protein), SOL-1, and GLR-1. Here, we use genetic and pharmacological perturbations along with rapid perfusion electrophysiological techniques to demonstrate that SOL-1 functions to slow the rate and limit the extent of receptor desensitization as well as to enhance the recovery from desensitization. We have also identified a SOL-1 homologue from Drosophila and show that Dro SOL1 has a conserved function in promoting C. elegans glutamate-gated currents. SOL-1 homologues may play critical roles in regulating glutamatergic neurotransmission in more complex nervous systems.
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Project description:Ionotropic glutamate receptors (iGluRs), a family of ligand-gated ion channels, are responsible for the majority of fast excitatory neurotransmission in the central nervous system. Within this family, different members serve distinct roles at glutamatergic synapses. Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate fast depolarization while N-methyl-D-aspartate (NMDA) receptors mediate the slower component of the excitatory postsynaptic potential. These disparate functions suggest alternate modes of regulation. In this work, we show that endogenous regulators of iGluRs have different abilities to bind to specific domains of NMDA NR1-1b and AMPA GluR2 subunits. We have previously shown that the sulfated neurosteroids pregnenolone sulfate and 3alpha-hydroxy-5beta-pregnan-20-one sulfate bind to the extracellular glutamate-binding core (S1S2) of the GluR2 subunit. Here we show that neither neurosteroid binds to the S1S2 domain of the NMDA NR1-1b subunit. This NR1-1b NMDA domain does, however, bind to the endogenous polyamines spermine and spermidine as well as Zn(II). Binding of the polyamines and Zn(II) to the S1S2 domain of the GluR2 subunit was not observed. This binding of Zn(II) and polyamines to the S1S2 domain of the NR1-1b subunit defines a new binding site for each of these modulators.
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Project description:Ionotropic glutamate receptors (iGluRs) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. We have characterized a family of iGluR-related genes in Drosophila, which we name ionotropic receptors (IRs). These receptors do not belong to the well-described kainate, AMPA, or NMDA classes of iGluRs, and they have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Misexpression of IRs in different olfactory neurons is sufficient to confer ectopic odor responsiveness. Together, these results lead us to propose that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues.
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Project description:Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here, we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed coreceptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into the use of IRs as genetically encoded chemical sensors.
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Project description:Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the central nervous system. iGluRs open their ion channels in response to binding of the neurotransmitter glutamate, rapidly depolarize the postsynaptic neuronal membrane, and initiate signal transduction. Recent studies using X-ray crystallography and cryo-electron microscopy have determined full-length iGluR structures that (1) uncover the receptor architecture in an unliganded, resting state, (2) reveal conformational changes produced by ligands in order to activate iGluRs, open their ion channels, and conduct ions, and (3) show how activated, glutamate-bound iGluRs can adopt a nonconducting desensitized state. These new findings, combined with the results of previous structural and functional experiments, kinetic and molecular modeling, mutagenesis, and biochemical analyses, provide new views on the structural mechanisms of iGluR gating.
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Project description:Ionotropic glutamate receptors from the AMPA and kainate subfamilies share many functional and structural features, but it is unclear whether this similarity extends to the molecular mechanisms underlying receptor desensitization. The current model for desensitization in AMPA receptors involves the rearrangement of dimers formed between subunit agonist binding domains. Key evidence for this has come from a single point mutant (from leucine to tyrosine) that abolished desensitization and that was shown to stabilize the binding domain dimer. However, the desensitization of kainate receptors appears to differ from that of AMPA receptors in several key respects. Although the kinetics of AMPA receptor gating and desensitization are consistent with channels formed from two dimers, similar evidence for the functional involvement of dimers has not been found in kainate receptors. Furthermore, despite the homolog of the nondesensitizing tyrosine in AMPA subunits also being a tyrosine in wild-type kainate subunits, these receptors desensitize rapidly and completely. Using mutagenesis based on the crystal structure of the glutamate receptor subunit GluR6 S1S2 domain in complex with domoate, we identified four residues neighboring this tyrosine that differ between AMPA and kainate subunits and that contribute to the different desensitization kinetics of these receptors. Detailed analysis of the effects of mutations at these sites confirms that there is in fact a common general mechanism for desensitization in non-NMDA receptors, dependent on the stability of the binding domain dimer interface, and reveals the existence of potential agonist-specific desensitization pathways.
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Project description:AMPA subtype ionotropic glutamate receptors (iGluRs) mediate the majority of fast neurotransmission across excitatory synapses in the central nervous system. Each AMPA receptor is composed of four multi-domain subunits that are organized into layers of two amino-terminal domain (ATD) dimers, two ligand-binding domain (LBD) dimers, transmembrane domains and carboxy-terminal domains. We introduced cysteine substitutions at the intersubunit interfaces of AMPA receptor subunit GluA2 and confirmed substituted cysteine crosslink formation by SDS-PAGE. The functional consequence of intersubunit crosslinks was assessed by recording GluA2-mediated currents in reducing and non-reducing conditions. Strong redox-dependent changes in GluA2-mediated currents were observed for cysteine substitutions at the LBD dimer-dimer interface but not at the ATD dimer-dimer interface. We conclude that during gating, LBD dimers undergo significant relative displacement, while ATD dimers either maintain their relative positioning, or their relative displacement has no appreciable effect on AMPA receptor function.
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Project description:Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.
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