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Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate., (© 2022. The Author(s).)

Delta receptors are members of the ionotropic glutamate receptor superfamily and form trans-synaptic connections by interacting with the extracellular scaffolding protein cerebellin-1 and presynaptic transmembrane protein neurexin-1β. Unlike other family members, however, direct agonist-gated ion channel activity has not been recorded in delta receptors. Here, we show that the GluD2 subtype of delta receptor forms cation-selective channels when bound to cerebellin-1 and neurexin-1β. Using fluorescence lifetime measurements and chemical cross-linking, we reveal that tight packing of the amino-terminal domains of GluD2 permits glycine- and d-serine–induced channel openings. Thus, cerebellin-1 and neurexin-1β act as biological cross-linkers to stabilize the extracellular domains of GluD2 receptors, allowing them to function as ionotropic excitatory neurotransmitter receptors in synapses.

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients., (U.S. Government work not protected by U.S. copyright.)

N -Methyl-D-aspartate receptors (NMDARs) are highly expressed in brain and play important roles in neurodevelopment and various neuropathologic conditions. Here, we describe a new phenotype in an individual associated with a novel de novo deleterious variant in GRIN1 (c.1595C>A, p.Pro532His). The clinical phenotype is characterized with developmental encephalopathy, striking stimulus-sensitive myoclonus, and frontal lobe and frontal white matter hypoplasia, with no apparent seizures detected. NMDARs that contained the P532H within the glycine-binding domain of GluN1 with either the GluN2A or GluN2B subunits were evaluated for changes in their pharmacological and biophysical properties, which surprisingly revealed only modest changes in glycine potency but a significant decrease in glutamate potency, an increase in sensitivity to endogenous zinc inhibition, a decrease in response to maximally effective concentrations of agonists, a shortened synaptic-like response time course, a decreased channel open probability, and a reduced receptor cell surface expression. Molecule dynamics simulations suggested that the variant can lead to additional interactions across the dimer interface in the agonist-binding domains, resulting in a more open GluN2 agonist-binding domain cleft, which was also confirmed by single-molecule fluorescence resonance energy transfer measurements. Based on the functional deficits identified, several positive modulators were evaluated to explore potential rescue pharmacology., Competing Interests: HY is PI on a research grant from Sage Therapeutics to Emory University School of Medicine. ST is PI on research grants from Janssen and Biogen to Emory University School of Medicine, is a member of the SAB for Sage Therapeutics and Eumentis, Inc., is co-founder of NeurOp Inc. and Agrithera Inc., and receives royalties for software. ST and HY are co-inventors on Emory-owned Intellectual Property that includes allosteric modulators of NMDA receptor function. SM owns shares in NeurOp. Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Tang, Bhatia, Xu, Paudyal, Liu, Kim, Song, XiangWei, Shaulsky, Myers, Dobyns, Jayaraman, Traynelis, Yuan and Bozarth.)

Many neurological disorders show an increased prevalence of GluA2-lacking, Ca 2+ -permeable AMPA receptors (CP-AMPARs), which dramatically alters synaptic function. However, the molecular mechanism underlying this distinct synaptic plasticity remains enigmatic. Here, we show that nerve injury potentiates postsynaptic, but not presynaptic, CP-AMPARs in the spinal dorsal horn via α2δ-1. Overexpressing α2δ-1, previously regarded as a Ca 2+ channel subunit, augments CP-AMPAR levels at the cell surface and synapse. Mechanistically, α2δ-1 physically interacts with both GluA1 and GluA2 via its C terminus, inhibits the GluA1/GluA2 heteromeric assembly, and increases GluA2 retention in the endoplasmic reticulum. Consequently, α2δ-1 diminishes the availability and synaptic expression of GluA1/GluA2 heterotetramers in the spinal cord in neuropathic pain. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-AMPAR complex fully restores the intracellular assembly and synaptic dominance of heteromeric GluA1/GluA2 receptors. Thus, α2δ-1 is a pivotal AMPAR-interacting protein that controls the subunit composition and Ca 2+ permeability of postsynaptic AMPARs., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Single molecule Förster Resonance Energy Transfer (smFRET) allows us to measure variation in distances between donor and acceptor fluorophores attached to a protein, providing the conformational landscape of the protein with respect to this specific distance. smFRET can be performed on freely diffusing molecules or on tethered molecules. Here, we describe the tethered method used to study ionotropic glutamate receptors, which allows us to track the changes in FRET as a function of time, thus providing information on the conformations sampled and kinetics of conformational changes in the millisecond to second time scale. Strategies for attaching fluorophores to the proteins, methods for acquiring and analyzing the smFRET trajectories, and limitations are discussed., (© 2021 Elsevier Inc. All rights reserved.)

N-methyl-D-aspartate (NMDA) receptors mediate synaptic excitatory signaling in the mammalian central nervous system by forming calcium-permeable transmembrane channels upon binding glutamate and coagonist glycine. Ca 2+ influx through NMDA receptors leads to channel inactivation through a process mediated by resident calmodulin bound to the intracellular C-terminal segment of the GluN1 subunit of the receptor. Using single-molecule FRET investigations, we show that in the presence of calcium-calmodulin, the distance across the two GluN1 subunits at the entrance of the first transmembrane segment is shorter and the bilobed cleft of the glycine-binding domain in GluN1 is more closed when bound to glycine and glutamate relative to what is observed in the presence of barium-calmodulin. Consistent with these observations, the glycine deactivation rate is slower in the presence of calcium-calmodulin. Taken together, these results show that the binding of calcium-calmodulin to the C-terminus has long-range allosteric effects on the extracellular segments of the receptor that may contribute to the calcium-dependent inactivation., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for investigating the structural dynamics of biological macromolecules. smFRET reveals the conformational landscape and dynamic changes of proteins by building on the static structures found using cryo-electron microscopy, x-ray crystallography, and other methods. Combining smFRET with static structures allows for a direct correlation between dynamic conformation and function. Here, we discuss the different experimental setups, fluorescence detection schemes, and data analysis strategies that enable the study of structural dynamics of glutamate signaling across various timescales. We illustrate the versatility of smFRET by highlighting studies of a wide range of questions, including the mechanism of activation and transport, the role of intrinsically disordered segments, and allostery and cooperativity between subunits in biological systems responsible for glutamate signaling., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Single-molecule fluorescence energy transfer methods allow us to determine the complete structural landscape between the donor and acceptor fluorophores introduced on the protein of interest. This method is particularly attractive to study ion channel proteins as single-molecule current recordings have been used to study the function of these proteins for several decades. Here we describe the smFRET method used to study glutamate receptors.

α2δ-1, commonly known as a voltage-activated Ca 2+ channel subunit, is a binding site of gabapentinoids used to treat neuropathic pain and epilepsy. However, it is unclear how α2δ-1 contributes to neuropathic pain and gabapentinoid actions. Here, we show that Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. Conversely, Cacna2d1 knockdown or ablation normalizes synaptic NMDAR activity increased by nerve injury. α2δ-1 forms a heteromeric complex with NMDARs in rodent and human spinal cords. The α2δ-1-NMDAR interaction predominantly occurs through the C terminus of α2δ-1 and promotes surface trafficking and synaptic targeting of NMDARs. Gabapentin or an α2δ-1 C terminus-interfering peptide normalizes NMDAR synaptic targeting and activity increased by nerve injury. Thus, α2δ-1 is an NMDAR-interacting protein that increases NMDAR synaptic delivery in neuropathic pain. Gabapentinoids reduce neuropathic pain by inhibiting forward trafficking of α2δ-1-NMDAR complexes., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fast excitatory transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs) associated with transmembrane AMPAR regulatory proteins (TARPs). At the high glutamate concentrations typically seen during synaptic transmission, TARPs slow receptor desensitization and enhance mean channel conductance. However, their influence on channels gated by low glutamate concentrations, as encountered during delayed transmitter clearance or synaptic spillover, is poorly understood. We report here that TARP γ-2 reduces the ability of low glutamate concentrations to cause AMPAR desensitization and enhances channel gating at low glutamate occupancy. Simulations show that, by shifting the balance between AMPAR activation and desensitization, TARPs can markedly facilitate the transduction of spillover-mediated synaptic signaling. Furthermore, the dual effects of TARPs can account for biphasic steady-state glutamate concentration-response curves-a phenomenon termed "autoinactivation," previously thought to reflect desensitization-mediated AMPAR/TARP dissociation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Acid-sensing ion channels (ASICs) are trimeric cation-selective ion channels activated by protons in the physiological range. Recent reports have revealed that postsynaptically localized ASICs contribute to the excitatory postsynaptic current by responding to the transient acidification of the synaptic cleft that accompanies neurotransmission. In response to such brief acidic transients, both recombinant and native ASICs show extremely rapid deactivation in outside-out patches when jumping from a pH 5 stimulus to a single resting pH of 8. Given that the resting pH of the synaptic cleft is highly dynamic and depends on recent synaptic activity, we explored the kinetics of ASIC1a and 1a/2a heteromers to such brief pH transients over a wider [H + ] range to approximate neuronal conditions better. Surprisingly, the deactivation of ASICs was steeply dependent on the pH, spanning nearly three orders of magnitude from extremely fast (<1 ms) at pH 8 to very slow (>300 ms) at pH 7. This study provides an example of a ligand-gated ion channel whose deactivation is sensitive to agonist concentrations that do not directly activate the receptor. Kinetic simulations and further mutagenesis provide evidence that ASICs show such steeply agonist-dependent deactivation because of strong cooperativity in proton binding. This capacity to signal across such a large synaptically relevant bandwidth enhances the response to small-amplitude acidifications likely to occur at the cleft and may provide ASICs with the ability to shape activity in response to the recent history of the synapse.

Key Points: Acid-sensing ion channels (ASICs) act as neurotransmitter receptors by responding to synaptic cleft acidification. We investigated how ASIC1a homomers and ASIC1a/2a heteromers respond to brief stimuli, jumping from pH 8.0 to 5.0, approximating the time course of neurotransmitter in the cleft. We find that ASICs deactivate surprisingly fast in response to such brief stimuli from pH 8.0 to 5.0, whereas they desensitize comparatively slowly to prolonged activation. The combination of unusually fast deactivation with slow desensitzation enables recombinant ASIC1a homomers and ASIC1a/2a heteromers, as well as native ASICs of sensory neurons, to follow trains of such brief pH 8.0 to 5.0 stimuli at high frequencies. This capacity for high-frequency signalling persists under a physiological pH of 7.4 with ASIC1a/2a heteromers, suggesting that they may sustain postsynaptic responses when other receptors desensitize., Abstract: The neurotransmitter-gated ion channels that underlie rapid synaptic transmission are often subjected to bursts of very brief neurotransmitter release at high frequencies. When challenged with such short duration high-frequency stimuli, neurotransmitter-gated ion channels generally exhibit the common response of desensitization. Recently, acid-sensing ion channels (ASICs) were shown to act as neurotransmitter-gated ion channels because postsynaptic ASICs can be activated by the transient acidification of the synaptic cleft accompanying neurotransmission. In the present study, we examined the responses of recombinant ASIC1a homomers, ASIC1a/2a heteromers and native ASICs from sensory neurons to 1 ms acidification stimuli, switching from pH 8.0 to 5.0, as either single pulses or trains of pulses at physiologically relevant frequencies. We found that ASIC deactivation is extremely fast and, in contrast to most other neurotransmitter-gated ion channels, ASICs show no desensitization during high-frequency stimulus trains under these conditions. We also found that accelerating ASIC desensitization by anion substitution can induce depression during high-frequency trains. When using a baseline physiological pH of 7.4, the ASIC1a responses were too small to reliably measure, presumably as a result of steady-state desensitization. However, ASIC1a/2 heteromers gave robust responses when using a baseline pH of 7.4 and were also able to sustain these responses during high-frequency stimulus trains. In conclusion, we report that the slow desensitization and fast deactivation of ASIC1a/2a heteromers enables them to sustain postsynaptic responses to bursts at high frequencies at a physiological pH that may desensitize other receptors., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Carboxylate groups incorporated at the position alpha to the keto carbonyl of alpha-keto amides 1 were photochemically cleaved in aqueous media to give carboxylic acids in 70-90% yields with quantum yields of 0.3. The cleavage coproducts were diastereomeric hemiacetals 2. Prompt release of acetate and gamma-aminobutyrate (GABA) in buffer was observed by difference FT-IR spectroscopy upon 355 nm laser flash photolysis. The time-constant for release of GABA was <30 ms. [reaction--see text]