A eukaryotic specific transmembrane segment is required for tetramerization in AMPA receptors

Most fast excitatory synaptic transmission in the nervous system is mediated by glutamate acting through ionotropic glutamate receptors (iGluRs). iGluRs (AMPA, kainate, and NMDA receptor subtypes) are tetrameric assemblies, formed as a dimer of dimers. Still, the mechanism underlying tetramerization-the necessary step for the formation of functional receptors that can be inserted into the plasma membrane-is unknown. All eukaryotic compared to prokaryotic iGluR subunits have an additional transmembrane segment, theM4segment, which positions the physiologically critical C-terminal domain on the cytoplasmic side of the membrane.AMPAreceptor (AMPAR) subunits lacking M4 do not express on the plasma membrane. Here, we show that these constructs are retained in the endoplasmic reticulum, the major cellular compartment mediating protein oligomerization. Using approaches to assay the native oligomeric state of AMPAR subunits, we find that subunits lacking M4 or containing single amino acid substitutions along an "interacting" face of the M4 helix that block surface expression no longer tetramerize in either homomeric or heteromeric assemblies. In contrast, subunit dimerization appears to be largely intact. These experiments define the M4 segment as a unique functional unit in AMPARs that is required for the critical dimer-to-tetramer transition. © 2013 the authors

Structure, function, and allosteric modulation of NMDA receptors

NMDA-type glutamate receptors are ligand-gated ion channels that mediate a Ca(2+)-permeable component of excitatory neurotransmission in the central nervous system (CNS). They are expressed throughout the CNS and play key physiological roles in synaptic function, such as synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the pathophysiology of several CNS disorders and more recently have been identified as a locus for disease-associated genomic variation. NMDA receptors exist as a diverse array of subtypes formed by variation in assembly of seven subunits (GluN1, GluN2A-D, and GluN3A-B) into tetrameric receptor complexes. These NMDA receptor subtypes show unique structural features that account for their distinct functional and pharmacological properties allowing precise tuning of their physiological roles. Here, we review the relationship between NMDA receptor structure and function with an emphasis on emerging atomic resolution structures, which begin to explain unique features of this receptor

Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule

N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system

Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis

RNA editing of kainate receptor subunits at the Q/R site determines their susceptibility to inhibition by cis-unsaturated fatty acids as well as block by cytoplasmic polyamines. Channels comprised of unedited (Q) subunits are strongly blocked by polyamines, but insensitive to fatty acids, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), whereas homomeric edited (R) channels resist polyamine block but are inhibited by AA and DHA. In the present study, we have analyzed fatty acid modulation of whole-cell currents mediated by homomeric recombinant GluK2 (formerly GluR6) channels with individual residues in the pore-loop, M1 and M3 transmembrane helices replaced by scanning mutagenesis. Our results define three abutting surfaces along the M1, M2, and M3 helices where gain-of-function substitutions render GluK2(Q) channels susceptible to fatty acid inhibition. In addition, we identify four locations in the M3 helix (F611, L614, S618, and T621) at the level of the central cavity where Arg substitution increases relative permeability to chloride and eliminates polyamine block. Remarkably, for two of these positions, L614R and S618R, exposure to fatty acids reduces the apparent chloride permeability and potentiates whole-cell currents ∼5 and 2.5-fold, respectively. Together, our results suggest that AA and DHA alter the orientation of M3 in the open state, depending on contacts at the interface between M1, M2, and M3. Moreover, our results demonstrate the importance of side chains within the central cavity in determining ionic selectivity and block by cytoplasmic polyamines despite the inverted orientation of GluK2 as compared with potassium channels and other pore-loop family members

Angiographic Features and Clinical Outcomes of Balloon Uncrossable Lesions during Chronic Total Occlusion Percutaneous Coronary Intervention

Background: Balloon uncrossable lesions are defined as lesions that cannot be crossed with a balloon after successful guidewire crossing. Methods: We analyzed the association between balloon uncrossable lesions and procedural outcomes of 8671 chronic total occlusions (CTOs) percutaneous coronary interventions (PCIs) performed between 2012 and 2022 at 41 centers. Results: The prevalence of balloon uncrossable lesions was 9.2%. The mean patient age was 64.2 ± 10 years and 80% were men. Patients with balloon uncrossable lesions were older (67.3 ± 9 vs. 63.9 ± 10, p \u3c 0.001) and more likely to have prior coronary artery bypass graft surgery (40% vs. 25%, p \u3c 0.001) and diabetes mellitus (50% vs. 42%, p \u3c 0.001) compared with patients who had balloon crossable lesions. In-stent restenosis (23% vs. 16%. p \u3c 0.001), moderate/severe calcification (68% vs. 40%, p \u3c 0.001), and moderate/severe proximal vessel tortuosity (36% vs. 25%, p \u3c 0.001) were more common in balloon uncrossable lesions. Procedure time (132 (90, 197) vs. 109 (71, 160) min, p \u3c 0.001) was longer and the air kerma radiation dose (2.55 (1.41, 4.23) vs. 1.97 (1.10, 3.40) min, p \u3c 0.001) was higher in balloon uncrossable lesions, while these lesions displayed lower technical (91% vs. 99%, p \u3c 0.001) and procedural (88% vs. 96%, p \u3c 0.001) success rates and higher major adverse cardiac event (MACE) rates (3.14% vs. 1.49%, p \u3c 0.001). Several techniques were required for balloon uncrossable lesions. Conclusion: In a contemporary, multicenter registry, 9.2% of the successfully crossed CTOs were initially balloon uncrossable. Balloon uncrossable lesions exhibited lower technical and procedural success rates and a higher risk of complications compared with balloon crossable lesions

Disease-associated missense mutations in GluN2B subunit alter NMDA receptor ligand binding and ion channel properties.

Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes. Here, we investigated several such mutations using a near-complete, hybrid 3D model of the human NMDAR and studied their consequences with kinetic modelling and electrophysiology. The mutants revealed reductions in glutamate potency; increased receptor desensitisation; and ablation of voltage-dependent Mg block. In addition, we provide new views on Mg and NMDA channel blocker binding sites. We demonstrate that these mutants have significant impact on excitatory transmission in developing neurons, revealing profound changes that could underlie their associated neurological disorders. Of note, the NMDAR channel mutant GluN2B unusually allowed Mg permeation, whereas nearby N615I reduced Ca permeability. By identifying the binding site for an NMDAR antagonist that is used in the clinic to rescue gain-of-function phenotypes, we show that drug binding may be modified by some GluN2B disease-causing mutations

dTip60 HAT Activity Controls Synaptic Bouton Expansion at the Drosophila Neuromuscular Junction

Background: Histone acetylation of chromatin plays a key role in promoting the dynamic transcriptional responses in neurons that influence the neuroplasticity linked to cognitive ability, yet the specific histone acetyltransferases (HATs) that create such epigenetic marks remain to be elucidated. Methods and Findings: Here we use the Drosophila neuromuscular junction (NMJ) as a well-characterized synapse model to identify HATs that control synaptic remodeling and structure. We show that the HAT dTip60 is concentrated both pre and post-synaptically within the NMJ. Presynaptic targeted reduction of dTip60 HAT activity causes a significant increase in synaptic bouton number that specifically affects type Is boutons. The excess boutons show a suppression of the active zone synaptic function marker bruchpilot, suggesting defects in neurotransmission function. Analysis of microtubule organization within these excess boutons using immunohistochemical staining to the microtubule associated protein futsch reveals a significant increase in the rearrangement of microtubule loop architecture that is required for bouton division. Moreover, a-tubulin acetylation levels of microtubules specifically extending into the terminal synaptic boutons are reduced in response to dTip60 HAT reduction. Conclusions: Our results are the first to demonstrate a causative role for the HAT dTip60 in the control of synaptic plasticity that is achieved, at least in part, via regulation of the synaptic microtubule cytoskeleton. These findings have implication

Global Chronic Total Occlusion Crossing Algorithm: JACC State-of-the-Art Review

The authors developed a global chronic total occlusion crossing algorithm following 10 steps: 1) dual angiography; 2) careful angiographic review focusing on proximal cap morphology, occlusion segment, distal vessel quality, and collateral circulation; 3) approaching proximal cap ambiguity using intravascular ultrasound, retrograde, and move-the-cap techniques; 4) approaching poor distal vessel quality using the retrograde approach and bifurcation at the distal cap by use of a dual-lumen catheter and intravascular ultrasound; 5) feasibility of retrograde crossing through grafts and septal and epicardial collateral vessels; 6) antegrade wiring strategies; 7) retrograde approach; 8) changing strategy when failing to achieve progress; 9) considering performing an investment procedure if crossing attempts fail; and 10) stopping when reaching high radiation or contrast dose or in case of long procedural time, occurrence of a serious complication, operator and patient fatigue, or lack of expertise or equipment. This algorithm can improve outcomes and expand discussion, research, and collaboration

Global Chronic Total Occlusion Crossing Algorithm

The authors developed a global chronic total occlusion crossing algorithm following 10 steps: 1) dual angiography; 2) careful angiographic review focusing on proximal cap morphology, occlusion segment, distal vessel quality, and collateral circulation; 3) approaching proximal cap ambiguity using intravascular ultrasound, retrograde, and move-the-cap techniques; 4) approaching poor distal vessel quality using the retrograde approach and bifurcation at the distal cap by use of a dual-lumen catheter and intravascular ultrasound; 5) feasibility of retrograde crossing through grafts and septal and epicardial collateral vessels; 6) antegrade wiring strategies; 7) retrograde approach; 8) changing strategy when failing to achieve progress; 9) considering performing an investment procedure if crossing attempts fail; and 10) stopping when reaching high radiation or contrast dose or in case of long procedural time, occurrence of a serious complication, operator and patient fatigue, or lack of expertise or equipment. This algorithm can improve outcomes and expand discussion, research, and collaboration.info:eu-repo/semantics/publishedVersio