@article {68293,
	title = {Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.},
	journal = {PLoS One},
	volume = {17},
	year = {2022},
	month = {2022 Aug 12},
	pages = {e0271799},
	abstract = {<p>Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.</p>
},
	keywords = {Animals, Cryoelectron Microscopy, Electron Microscope Tomography, Lasers, Mice, Microscopy, Confocal, Synapses, Synaptosomes},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0271799},
	author = {Gogoi, Prerana and Shiozaki, Momoko and Gouaux, Eric}
}
@article {2317,
	title = {Mechanism of positive allosteric modulators acting on AMPA receptors.},
	journal = {The Journal of Neuroscience: The Official Journal of the Society for Neuroscience},
	volume = {25},
	year = {2005},
	month = {2005 Sep 28},
	pages = {9027-36},
	abstract = {<p>Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer{\textquoteright}s disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1,3-oxazino benzo-1,4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the "hinge" in the ligand-binding core "clamshell" that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.</p>
},
	keywords = {Allosteric Regulation, AMPA, Benzothiadiazines, Binding Sites, Cell Line, Crystallography, Dimerization, Drug Synergism, Electric Conductivity, Humans, Ligands, Molecular Conformation, Oxazines, Patch-Clamp Techniques, Protein Structure, Pyrrolidinones, Receptors, Tertiary, Time Factors},
	issn = {1529-2401},
	doi = {10.3389/fnana.2010.00147},
	author = {Jin, Rongsheng and Clark, Suzanne and Weeks, Autumn M and Dudman, Joshua T and Gouaux, Eric and Partin, Kathryn M}
}
