Single-particle cryo-EM at atomic resolution.

The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the ß3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

Alternative Splicing of the Flip/Flop Cassette and TARP Auxiliary Subunits Engage in a Privileged Relationship That Fine-Tunes AMPA Receptor Gating.

Alternative splicing of AMPA-type glutamate receptors (AMPARs) and allosteric modulation by auxiliary subunits, such as transmembrane AMPAR regulatory proteins (TARPs), are two important mechanisms that regulate the time course of glutamatergic neurotransmission. Prior work has shown that alternative splicing of the flip/flop cassette profoundly regulates TARP γ2 modulation, where flip receptor gating exhibits robust sensitivity to TARPs while flop isoforms are relatively insensitive to TARP modulation. Whether this splice variant-specific regulation extends to other auxiliary subunit families, such as cornichons (CNIHs), GSG1L, or CKAMPs, remains unknown. Here, we demonstrate that CNIH-3 modulation is unaffected by AMPAR alternative splicing due to inherent differences in how CNIH-3 and TARP γ2 modify channel gating. CNIH-3 slows receptor deactivation from the outset of current decay, consistent with structural evidence showing its point of contact at the level of the pore. In contrast, TARP γ2 acts via the KGK site of the ligand-binding domain (LBD) to slow the onset of desensitization. Although GSG1L and CKAMP44 primarily slow recovery from desensitization, their effects on channel gating are unaffected by alternative splicing, further underlining that structural events leading to the onset and recovery from desensitization are separable. Together, this work establishes that alternative splicing and TARP auxiliary subunits form a unique partnership that governs fast glutamatergic signaling at central synapses. Since proteomic studies suggest that all native AMPARs co-assemble with at least two TARPs, allosteric coupling between the flip/flop cassette and TARPs may represent a common design element in all AMPAR complexes of the mammalian brain.SIGNIFICANCE STATEMENT All fast excitatory neurotransmission in the mammalian brain is mediated by AMPA-type glutamate receptors (AMPARs). The time course of AMPAR gating can be regulated by two distinct mechanisms: alternative splicing of the flip/flop cassette and association with auxiliary subunits. Although these regulatory mechanisms have been well studied individually, it is not clear whether alternative splicing impacts auxiliary protein modulation of AMPARs. Here, we compare the four main families of AMPAR auxiliary subunits, transmembrane AMPAR regulatory proteins (TARPs; γ2), cornichons (CNIH-3), GSG1L and CKAMPs (CKAMP44), and find a privileged relationship between TARPs and the flip/flop cassette that is not shared by others. The flop cassette acts as a master switch to override TARP action, and this coupling represents a way to fine-tune AMPAR signaling.

Functional Validation of Heteromeric Kainate Receptor Models.

Kainate receptors require the presence of external ions for gating. Most work thus far has been performed on homomeric GluK2 but, in vivo, kainate receptors are likely heterotetramers. Agonists bind to the ligand-binding domain (LBD) which is arranged as a dimer of dimers as exemplified in homomeric structures, but no high-resolution structure currently exists of heteromeric kainate receptors. In a full-length heterotetramer, the LBDs could potentially be arranged either as a GluK2 homomer alongside a GluK5 homomer or as two GluK2/K5 heterodimers. We have constructed models of the LBD dimers based on the GluK2 LBD crystal structures and investigated their stability with molecular dynamics simulations. We have then used the models to make predictions about the functional behavior of the full-length GluK2/K5 receptor, which we confirmed via electrophysiological recordings. A key prediction and observation is that lithium ions bind to the dimer interface of GluK2/K5 heteromers and slow their desensitization.

α6-Containing GABAA Receptors Are the Principal Mediators of Inhibitory Synapse Strengthening by Insulin in Cerebellar Granule Cells.

Activity-dependent strengthening of central synapses is a key factor driving neuronal circuit behavior in the vertebrate CNS. At fast inhibitory synapses, strengthening is thought to occur by increasing the number of GABAA receptors (GABARs) of the same subunit composition to preexisting synapses. Here, we show that strengthening of mouse cerebellar granule cell GABAergic synapses occurs by a different mechanism. Specifically, we show that the neuropeptide hormone, insulin, strengthens inhibitory synapses by recruiting α6-containing GABARs rather than accumulating more α1-containing receptors that are resident to the synapse. Because α6-receptors are targeted to functionally distinct postsynaptic sites from α1-receptors, we conclude that only a subset of all inhibitory synapses are strengthened. Together with our recent findings on stellate cells, we propose a general mechanism by which mature inhibitory synapses are strengthened. In this scenario, α1-GABARs resident to inhibitory synapses form the hardwiring of neuronal circuits with receptors of a different composition fulfilling a fundamental, but unappreciated, role in synapse strengthening.

Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.

KEY POINTS: Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion-channel block by facilitating blocker permeation. Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α-helical region. Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism. Our findings have broad implications for work on polyamine block of other cation-selective ion channels. ABSTRACT: Channel block and permeation by cytoplasmic polyamines is a common feature of many cation-selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α-helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation-selective ion channels.

Hydrogen peroxide increases GABAA receptor-mediated tonic current in hippocampal neurons.

Hydrogen peroxide (H2O2), a key reactive oxygen species, is produced at low levels during normal cellular metabolism and at higher concentrations under pathological conditions such as ischemia-reperfusion injury. The mechanisms by which H2O2 contributes to physiological and pathological processes in the brain remain poorly understood. Inhibitory GABA type A (GABAA) receptors critically regulate brain function by generating tonic and synaptic currents; however, it remains unknown whether H2O2 directly modulates GABAA receptor function. Here, we performed patch-clamp recordings, together with pharmacological and genetic approaches, to investigate the effects of H2O2 on GABAA receptor-mediated tonic and synaptic currents recorded in cultured mouse hippocampal neurons and CA1 pyramidal neurons in hippocampal slices. We found that H2O2 caused a dramatic increase in tonic current, whereas synaptic currents were unaffected. This increase in tonic current resulted from an extracellular oxidative reaction, which increased the potency of GABA, but only when GABAA receptors were activated by low concentrations of GABA. Oxygen-glucose deprivation, which produces high endogenous levels of H2O2, similarly increased the tonic current. These results suggest that GABAA receptor-mediated tonic current, which is potentiated by H2O2, might contribute to H2O2-induced brain dysfunction.

Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.

Most ligand- and voltage-gated ion channels assemble as signaling complexes consisting of pore-forming and auxiliary subunits. In the mammalian brain, AMPA-type ionotropic glutamate receptors (AMPARs) coassemble with several families of auxiliary subunits that regulate channel gating as well as ion channel block and permeation. Previous work has shown that auxiliary proteins stargazin (or γ2) and cornichon-3 (CNIH-3) attenuate the cytoplasmic polyamine channel block of AMPARs, although the underlying mechanism has yet to be established. Here, we show that γ2 and CNIH-3 relieve channel block by enhancing the rate of blocker permeation. Surprisingly, the relative permeability of the polyamine spermine (Spm) through the pore of the AMPAR-γ2 or -CNIH-3 complexes is considerably more than AMPARs expressed alone. Spm permeability is comparable to that of Na+ for the GluA2-γ2 complex and four times greater than Na+ with GluA2 + CNIH-3. A modified model of permeant channel block fully accounts for both the voltage- and time-dependent nature of Spm block. Estimates of block rate constants reveal that auxiliary subunits do not attenuate block by shifting the location of the block site within the membrane electric field, and they do not affect the blocker's ability to reach it. Instead, γ2 and CNIH-3 relieve channel block by facilitating the blocker's exit rates from the open channel. From a physiological perspective, the relief of channel block exerted by γ2 and CNIH-3 ensures that there is unfettered signaling by AMPARs at glutamatergic synapses. Moreover, the pronounced ability of AMPARs to transport polyamines may have an unexpected role in regulating cellular polyamine levels.

Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (Ki = 4 µM). In a functional assay, 1b displayed full antagonist activity with IC50 = 6 ± 2 µM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

Binding mode of an α-amino acid-linked quinoxaline-2,3-dione analogue at glutamate receptor subtype GluK1.

Two α-amino acid-functionalized quinoxalines, 1a (CNG-10301) and 1b (CNG-10300), of a quinoxaline moiety coupled to an amino acid moiety were designed, synthesized, and characterized pharmacologically. While 1a displayed low affinity at native AMPA, KA, and NMDA receptors, and at homomeric GluK1,3 receptors, the affinity for GluK2 was in the midmicromolar range (Ki = 136 µM), 1b displayed low to midmicromolar range binding affinity at all the iGluRs (Ki = 9-126 µM). In functional experiments (outside-out patches excised from transfected HEK293T cells), 100 µM 1a partially blocked GluK1 (33% peak response), while GluK2 was unaffected (96% peak response). Furthermore, 1a was shown not to be an agonist at GluK1 and GluK2 at 100 µM. On the other hand, 100 µM 1b fully antagonized GluK1 (8% peak response) but only partially blocked GluK2 (33% peak response). An X-ray structure at 2.3 Å resolution of 1b in the GluK1-LBD (ligand-binding domain) disclosed an unexpected binding mode compared to the predictions made during the design phase; the quinoxaline moiety remains to act as an amino acid bioisostere, but the amino acid moiety is oriented into a new area within the GluK1 receptor. The structure of the GluK1-LBD with 1b showed a large variation in domain openings of the three molecules from 25° to 49°, demonstrating that the GluK1-LBD is capable of undergoing major domain movements.

Mitochondrial reactive oxygen species regulate the strength of inhibitory GABA-mediated synaptic transmission.

Neuronal communication imposes a heavy metabolic burden in maintaining ionic gradients essential for action potential firing and synaptic signalling. Although cellular metabolism is known to regulate excitatory neurotransmission, it is still unclear whether the brain's energy supply affects inhibitory signalling. Here we show that mitochondrial-derived reactive oxygen species (mROS) regulate the strength of postsynaptic GABA(A) receptors at inhibitory synapses of cerebellar stellate cells. Inhibition is strengthened through a mechanism that selectively recruits α3-containing GABA(A) receptors into synapses with no discernible effect on resident α1-containing receptors. Since mROS promotes the emergence of postsynaptic events with unique kinetic properties, we conclude that newly recruited α3-containing GABA(A) receptors are activated by neurotransmitter released onto discrete postsynaptic sites. Although traditionally associated with oxidative stress in neurodegenerative disease, our data identify mROS as a putative homeostatic signalling molecule coupling cellular metabolism to the strength of inhibitory transmission.