Structural insights into human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity, that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. This is the first characterization of molecular features of EAAT2 and the selective inhibition mechanism, underlying structure-based drug design for EAAT2.

A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers

There is growing interest in developing biologics due to their high target selectivity. The G protein–coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known. Here, we describe a nanobody that specifically and fully activates homodimeric human mGlu4 receptors. Molecular modeling and mutagenesis studies revealed that the nanobody acts by stabilizing the closed active state of the glutamate binding domain by interacting with both lobes. In contrast, this nanobody does not activate the heterodimeric mGlu2-4 but acts as a pure positive allosteric modulator. These data further reveal how an antibody can fully activate a class C receptor and bring further evidence that nanobodies represent an alternative way to specifically control mGlu receptor subtypes.

Study Of The Neuroprotective Properties Of Biologically Active Compounds

The works show that, using fluorescent probes, it was used to study the effect of PC-8 on changes in the dynamics of the intracellular Ca2+ content in synaptosomes of the rat brain, depending on the site of the binding of glutamate on calcium channels by a specific mediator with glutamate. To measure the amount of cytosolic Ca2+ synaptosomes, we calculated using the Grinkevich equation. It has been shown that polyphenol PC-8 binds to the glutamate-binding site of NMDA receptors, so that the conductance for Ca2+ ions is reduced through a channel blocking the effect of polyphenol PC-8 can be explained. due to its binding to the competing action of the site, the binding of glutamate to NMDA receptors.

The Mechanism Of Action Of Polyphenol On Changes In The Dynamics Of Calcium In The Synaptosomes Of The Rat Brain Against The Background Of Glutamate

The manuscript shows a short data used using fluorescent probes to study the effect of polyphenol PС-7 on changes in the dynamics of intracellular Ca2+ content in rat brain synaptosomes, depending on the site of glutamate binding on calcium channels by a specific mediator with glutamate. To measure the amount of cytosolic Ca2+ synaptosomes, we calculated using the Grinkevich equation. It has been shown that polyphenol PС-7 binds to the β1-subunit of the voltage-gated calcium channel and allosterically changes its conformation so that the conductivity for Ca2+ ions increases through the channel, the blocking effect of polyphenol PС-7 can be explained by its binding to voltage-gated calcium channels and activating them.

Cross-subunit interactions that stabilize open states mediate gating in NMDA receptors

NMDA receptors are excitatory channels with critical functions in the physiology of central synapses. Their activation reaction proceeds as a series of kinetically distinguishable, reversible steps, whose structural bases are currently under investigation. Very likely, the earliest steps include glutamate binding to glycine-bound receptors and subsequent constriction of the ligand-binding domain. Later, three short linkers transduce this movement to open the gate by mechanical pulling on transmembrane helices. Here, we used molecular and kinetic simulations and double-mutant cycle analyses to show that a direct chemical interaction between GluN1-I642 (on M3 helix) and GluN2A-L550 (on L1-M1 linker) stabilizes receptors after they have opened and thus represents one of the structural changes that occur late in the activation reaction. This native interaction extends the current decay, and its absence causes deficits in charge transfer by GluN1-I642L, a pathogenic human variant.

Alternative splicing of GluN1 gates glycine-primed internalization of NMDA receptors

SummaryN-methyl-D-aspartate receptors (NMDARs), a principal subtype of excitatory neurotransmitter receptor, are composed as tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Gating of the NMDARs requires binding of four co-agonist molecules, but the receptors can signal non-ionotropically through binding of glycine, alone, to its cognate site on GluN1. A consequence of this signalling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit is not a singular molecular species in the CNS, rather there are 8 alternatively spliced isoforms of this subunit produced by including or excluding the N1 and the C1, C2 or C2’ polypeptide cassettes. Whether alternative splicing affects glycine priming signalling is unknown. Here, using recombinant NMDARs expressed heterologously we discovered that glycine priming of NMDARs critically depends on alternative splicing: the four splice isoforms lacking the N1 cassette, encoded in exon 5, are primed by glycine whereas glycine priming is blocked in the four splice variants containing the N1 cassette. On the other hand, the C-terminal cassettes – C1, C2 or C2’ – had no effect on glycine priming signalling. Nor was glycine priming affected by the GluN2 subunit in the receptor. In wild-type mice we found that glycine primed internalization of synaptic NMDARs in CA1 hippocampal pyramidal neurons. With mice we engineered such that GluN1 obligatorily contained the N1 cassette, glycine did not prime synaptic NMDARs in pyramidal neurons. In contrast to pyramidal neurons, we discovered that in wild-type mice, synaptic NMDARs in CA1 inhibitory interneurons were resistant to glycine priming. But we recapitulated glycine priming in inhibitory interneurons in mice engineered such that GluN1 obligatorily lacked the N1 cassette. Our findings reveal a previously unsuspected molecular function for alternative splicing of GluN1 in controlling non-ionotropic signalling of NMDAR by glycine and the consequential cell surface dynamics of the receptors.

Development of highly selective fluorescent sensors for detection of glycolipids in vesicles and visualization of glutamate for neuronal imaging

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI-COLUMBIA AT REQUEST OF AUTHOR.] Fluorescent sensors are very useful tools for exploring chemical biology and advanced medical research. Herein, we propose four different fluorescent sensor systems for the recognition of some important biological molecules. The first sensor system is a multi-component fluorescent sensor complex for the sensing of glycolipids. The glycolipid sensor system is a novel design that takes advantage of supramolecular self-assembly. Results show that it can bind with both the sugar headgroup and hydrocarbon tail of glycolipids, and turn on the fluorescence of the sensor system. The second sensor is a cell-impermeable fluorescent sensor system for the recognition and extraction of glycolipids from vesicles. To avoid the fluorescence enhancement caused by the hydrophobic effect from cell membrane, we designed a series of cell-impermeable sensor complexes. In addition, these complexes were fully explored by vesicle studies. Another fluorescent sensor is NS600 which was developed for detecting and imaging glutamate in neurons. This sensor system that utilizes a nucleophilic aromatic substitution for glutamate binding, and produces a 270-fold fluorescence enchantment upon glutamate binding. Also, it overcomes drawbacks of previous glutamate sensors including low signal response and poor sensitivity. It enables a clear and accurate visualization of glutamate in cultural neurons. The last sensor is NS570, a cell-impermeable glutamate sensor which could be loaded into synaptic vesicles by vesicle cycling. This sensor is a reversible chemical sensor that gives a 2600-fold fluorescence enhancement upon the titration with glutamate and can be used to monitor the release of neuronal glutamate in real time.

Ion transport and regulation in a synaptic vesicle glutamate transporter

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo–electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.