Central Vestibule Research Articles

Preliminary Report on the Twenty-Second Excavation Season of the Archaeological Expedition to Wad Ben Naga

The twenty-second excavation season of the Archaeological Expedition to Wad Ben Naga focused on the continued exploration of the so-called Isis Temple (WBN 300). The works covered the south-eastern part of the hypostyle hall (WBN 301), a part of the central vestibule (WBN 302) including its front entrance, the entire southern vestibule (WBN 310), and a large part of the southern corridor (WBN 312). The excavation in the hypostyle hall yielded most notably many fragments of columns and a complete abacus with relief decoration, which allowed for partial reconstruction of their decorative scheme. There were also many pieces of modelled and painted plaster fragments apparently coming from the southern tower of the pylon and depicting King Natakamani and the god Horus. The stone-built entrance from the hypostyle hall to the central vestibule was found to be covered with relief depictions of King Natakamani and Queen Amanitore and Egyptian hieroglyphic texts. The southern vestibule and the southern corridor were recognised to have had painted decoration, which was especially well preserved in the southern corridor. Besides the paintings, a stairway leading to the roof of the building and a later burial of a non-adult individual were located there as well.

Structural basis of ketamine action on human NMDA receptors.

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.

In vitro and in silico characterization of the inhibition of Kir4.1 channels by aminoglycoside antibiotics.

Aminoglycoside antibiotics are positively charged molecules that are known to inhibit several ion channels. In this study, we have shown that aminoglycosides also inhibit the activity of Kir4.1 channels. Aminoglycosides inhibit Kir4.1 channels by a pore-blocking mechanism, plugging the central vestibule of the channel. Patch-clamp recordings were made in HEK-293 cells transiently expressing Kir4.1 channels to analyse the effects of gentamicin, neomycin and kanamycin. In silico modelling followed by mutagenesis were realized to identify the residues critical for aminoglycosides binding to Kir4.1. Aminoglycoside antibiotics block Kir4.1 channels in a concentration- and voltage-dependent manner, getting access to the protein from the intracellular side of the plasma membrane. Aminoglycosides block Ki4.1 with a rank order of potency as follows: gentamicin ˃ neomycin ˃ kanamycin. The residues T128 and principally E158, facing the central cavity of Kir4.1, are important structural determinants for aminoglycosides binding to the channel, as determined by our in silico modelling and confirmed by mutagenesis experiments. Kir4.1 channels are also target of aminoglycoside antibiotics, which could affect potassium transport in several tissues.

Sevanol and Its Analogues: Chemical Synthesis, Biological Effects and Molecular Docking.

Among acid-sensing ion channels (ASICs), ASIC1a and ASIC3 subunits are the most widespread and prevalent in physiological and pathophysiological conditions. They participate in synaptic plasticity, learning and memory, as well as the perception of inflammatory and neurological pain, making these channels attractive pharmacological targets. Sevanol, a natural lignan isolated from Thymus armeniacus, inhibits the activity of ASIC1a and ASIC3 isoforms, and has a significant analgesic and anti-inflammatory effect. In this work, we described the efficient chemical synthesis scheme of sevanol and its analogues, which allows us to analyze the structure–activity relationships of the different parts of this molecule. We found that the inhibitory activity of sevanol and its analogues on ASIC1a and ASIC3 channels depends on the number and availability of the carboxyl groups of the molecule. At the structural level, we predicted the presence of a sevanol binding site based on the presence of molecular docking in the central vestibule of the ASIC1a channel. We predicted that this site could also be occupied in part by the FRRF-amide peptide, and the competition assay of sevanol with this peptide confirmed this prediction. The intravenous (i.v.), intranasal (i.n.) and, especially, oral (p.o.) administration of synthetic sevanol in animal models produced significant analgesic and anti-inflammatory effects. Both non-invasive methods of sevanol administration (i.n. and p.o.) showed greater efficacy than the invasive (i.v.) method, thus opening new horizons for medicinal uses of sevanol.

Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7

P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body β-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels.

Mutations in the palm domain disrupt modulation of acid-sensing ion channel 1a currents by neuropeptides

Modulation by neuropeptides enhances several functions of acid-sensing ion channels (ASICs), such as pain sensation and acid-induced neuronal injury. The acid-induced opening of ASICs is transient, because of a rapid desensitization. Neuropeptides containing an Arg-Phe-amide motif affect ASIC desensitization and allow continuous activity of ASICs. In spite of the importance of the sustained ASIC activity during prolonged acidification, the molecular mechanisms of ASIC modulation by neuropeptides is only poorly understood. To identify the FRRFa (Phe-Arg-Arg-Phe-amide) binding site on ASIC1a, we carried out an in silico docking analysis and verified functionally the docking predictions. The docking experiments indicated three possible binding pockets, located (1) in the acidic pocket between the thumb, finger, β-ball and palm domains, (2) in a pocket at the bottom of the thumb domain, and (3) in the central vestibule along with the connected side cavities. Functional measurements of mutant ASIC1a confirmed the importance of residues of the lower palm, which encloses the central vestibule and its side cavities, for the FRRFa effects. The combined docking and functional experiments strongly suggest that FRRFa binds to the central vestibule and its side cavities to change ASIC desensitization.

Ginsenosides Act As Positive Modulators of P2X4 Receptors

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP− or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.

Divalent cation and chloride ion sites of chicken acid sensing ion channel 1a elucidated by x-ray crystallography

Acid sensing ion channels (ASICs) are proton-gated ion channels that are members of the degenerin/epithelial sodium channel superfamily and are expressed throughout central and peripheral nervous systems. ASICs have been implicated in multiple physiological processes and are subject to numerous forms of endogenous and exogenous regulation that include modulation by Ca2+ and Cl- ions. However, the mapping of ion binding sites as well as a structure-based understanding of the mechanisms underlying ionic modulation of ASICs have remained elusive. Here we present ion binding sites of chicken ASIC1a in resting and desensitized states at high and low pH, respectively, determined by anomalous diffraction x-ray crystallography. The acidic pocket serves as a nexus for divalent cation binding at both low and high pH, while we observe divalent cation binding within the central vestibule on the resting channel at high pH only. Moreover, neutralization of residues positioned to coordinate divalent cations via individual and combined Glu to Gln substitutions reduced, but did not extinguish, modulation of proton-dependent gating by Ca2+. Additionally, we demonstrate that anion binding at the canonical thumb domain site is state-dependent and present a previously undetected anion site at the mouth of the extracellular fenestrations on the resting channel. Our results map anion and cation sites on ASICs across multiple functional states, informing possible mechanisms of modulation and providing a blueprint for the design of therapeutics targeting ASICs.

Quercetin inhibits acid-sensing ion channels through a putative binding site in the central vestibular region

Acid-sensing ion channels (ASICs) are associated with many pathophysiological processes, such as neuronal death during ischemic stroke, epileptic seizure and nociception. However, there is a dearth of ASIC-specific therapeutic blockers. Here we report that quercetin, a plant flavonoid, which is known for its neuroprotective effect, reversibly inhibits homomeric rat ASIC1a, ASIC2a and ASIC3 with an IC50 of about 2µM. Also, quercetin prevents low pH-induced intracellular calcium rise and cell death in HEK-293 cells, which have endogenous expression of ASIC1a and 2a. The inhibitory effect of quercetin on ASICs is not due to membrane perturbation, as it did not have any effect on other channels, like NMDA receptor, GABAA receptor and P2X4 receptor. Unlike quercetin, another flavonoid resveratrol had no effect on ASIC1a. Computational analysis revealed that quercetin binds to the channel in a cavity at the central vestibule, lined by several charged residues like Q276, R369, E373 and E416 in ASIC1a. Mutation of Arg369 to Ala or Glu416 to Gln abolished the inhibitory effect of quercetin on rat ASIC1a completely, while Glu373 to Gln showed reduced sensitivity. Our results raise the possibility of using quercetin for targeting ASICs in vivo.

Gating Mechanism and Movements in Acid Sensing Ion Channel 1A

Acid Sensing Ion Channels (ASICs) are proton sensitive ion channels found in central and peripheral nervous system. They are involved in a variety of physiological processes including sensory perception, synaptic transmission and nociception. It is also involved in pathological processes like stroke and hypoxia leading to cell death. Thus understanding the mechanism of working of these ion channels are of importance in order to be able to design modulators. Here we present results from a combination of biophysical, molecular, functional and computational studies that looks at various aspects of ASIC functions. Earlier studies pointed towards possible involvement of carboxylate residues in the extracellular domain, in pH sensing. Our results indicate that there are three carboxylate pairs that are involved in gating. The carboxylates upon proton binding, induce a conformational change that causes the finger and thumb domains of ASIC come closer to each other. Our results indicate that these carboxylate pairs and the conformational change are key factors that mediate gating of the ion channel. We then proceeded to study the inter subunit conformational changes that might be involved in desensitization process. Biophysical investigations indicate that the subunits move away from each other upon desensitization. This movement is not global between the subunits. The segments closer to the central vestibule stay relatively in the same position whereas the residues towards the periphery of the ion channel moves away from each other more prominently. These results show that the ion channel undergoes a lateral flowering-like motion during desensitization in comparison with the resting state of the ion channel.

NMDA receptor structures reveal subunit arrangement and pore architecture.

SummaryN-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present x-ray crystal structures of the GluN1/GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino terminal and ligand binding domains. The transmembrane domains harbor a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ~2-fold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating

P2X receptors are ATP-gated cation channels consisting of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. Here we used cysteine and alanine scanning mutagenesis of the rat P2X4R receptor V47–V61 and K326–N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closed-open-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, I332, I333, T335, I337, and N338 in channels undergoing a transition from an open-to-desensitized state; residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating was stimulatory in all reactive V47–V61 mutants and inhibitory in the majority of reactive K326–N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition were located within the lumen of the extracellular vestibule and toward the central vestibule; however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region.

Conformational Dynamics inside Amino-Terminal Disease Hotspot of Ryanodine Receptor

The N-terminal region of both skeletal and cardiac ryanodine receptor is a disease mutation hotspot. Recently, a crystal structure of the RyR1 fragment (residues 1-559) was solved. This N-terminal structure contains three separate domains, A, B, and C, and was docked into a central vestibule in a full-length RyR1 cryo-EM map. Here, we reconstructed three-dimensional cryo-EM structures of two GFP-tagged RyR2s with GFP inserted after residue Glu-310 and Ser-437, respectively. The structures of RyR2E310-GFP and RyR2S437-GFP displayed an extra mass on domain B and C, directly validating the predicted docking model. Next, we revealed domain movements in molecular dynamics flexible fitting models in both the closed and open state cryo-EM maps. To further probe the conformational changes, we generated FRET pairs by inserting CFP or YFP in two selected domains, FRET studies of three dual-insertion pairs and three co-expressed single-insertion pairs showed the dynamic structural changes within the N-terminal domains.

Molecular determinants of desensitization in an ENaC/degenerin channel

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Å above the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

Conformational Changes in the Lower Palm Domain of ASIC1a Contribute to Desensitization and RFamide Modulation

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel that contributes to fear and pain as well as neuronal damage following persistent cerebral acidosis. Neuropeptides can affect acid-induced neuronal injury by altering ASIC1a inactivation and/or steady-state desensitization. Yet, exactly how ASIC1a inactivation and desensitization occur or are modulated by peptides is not completely understood. We found that regions of the extracellular palm domain and the β11-12 linker are important for inactivation and steady-state desensitization of ASIC1a. The single amino acid substitutions L280C and L415C dramatically enhanced the rate of inactivation and altered the pH-dependence of steady-state desensitization. Further, the use of methanethiosulfonate (MTS) reagents suggests that the lower palm region (L280C) undergoes a conformational change when ASIC1a transitions from closed to desensitized. We determined that L280C also displays an altered response to the RFamide peptide, FRRFamide. Further, the presence of FRRFamide limited MTS modification of L280C. Together, these results indicate a potential role of the lower palm domain in peptide modulation and suggest RFamide-related peptides promote conformational changes within this region. These data provide empirical support for the idea that L280, and likely this region of the central vestibule, is intimately involved in channel inactivation and desensitization.

Modeling a Ryanodine Receptor Amino-Terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

Ryanodine receptors (RyRs) form a class of intracellular calcium release channels in various excitable tissues and cells such as muscles and neurons. They are the major cellular mediators of the release of calcium ions from the sarcoplasmic reticulum, an essential step in muscle excitation-contraction coupling. Several crystal structures of skeletal muscle RyR1 peptide fragments have been solved, but these cover less than 15% of the full-length RyR1 sequence. In the present study, we obtain pseudo-atomic models for RyR fragments, consisting of residues 850-1,056 in rabbit RyR1 or residues 861-1,067 in mouse RyR2, by combining modeling techniques with sub-nanometer resolution cryo-electron microscopy (cryo-EM) maps. These fragments dock into a domain that connects the central vestibule and corner clamp region of RyR, with a good match between the secondary structure elements in the cryo-EM map and the pseudo-atomic models, and also consistent with our previous results of 3D cryo-EM RyR-GFP mapping and FRET measurement between RyR and FKBP. A combined model of the RyR fragment and FKBP docked into the cryo-EM map suggests that the fragment is positioned adjacent to the FKBP binding site. Its predicted binding interface with FKBP consists of primarily electrostatic contacts and contains several disease-associated mutations. A dynamic interaction between the fragment and a RyR phosphorylation domain, characterized using FRET data, also support the structural predictions of the pseudo-atomic models.

Modeling a Ryanodine Receptor N-terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

Ryanodine receptors (RyRs) form a class of intracellular calcium release channels in various excitable tissues and cells such as muscles and neurons. They are the major cellular mediators of the release of calcium ions from the sarcoplasmic reticulum, an essential step in muscle excitation-contraction coupling. Several crystal structures of skeletal muscle RyR1 peptide fragments have been solved, but these cover less than 15% of the full-length RyR1 sequence. In this study, by combining modeling techniques with sub-nanometer resolution cryo-electron microscopy (cryo-EM) maps, we obtained pseudo-atomic models for RyR fragments consisting of residues 850-1,056 in rabbit RyR1 or residues 861-1,067 in mouse RyR2. These fragments are docked into a domain that connects the central vestibule and corner clamp region of RyR, resulting in a good match of the secondary structure elements in the cryo-EM map and the pseudo-atomic models, which is also consistent with our previous mappings of GFP insertions by cryo-EM and with FRET measurements involving RyR and FK506-binding protein (FKBP). A combined model of the RyR fragment and FKBP docked into the cryo-EM map suggests that the fragment is positioned adjacent to the FKBP-binding site. Its predicted binding interface with FKBP consists primarily of electrostatic contacts and contains several disease-associated mutations. A dynamic interaction between the fragment and an RyR phosphorylation domain, characterized by FRET experiments, also supports the structural predictions of the pseudo-atomic models.

Domain Architecture of a Calcium-Permeable AMPA Receptor in a Ligand-Free Conformation

Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation.

Structure of the Acid-sensing ion channel 1 in complex with the gating modifier Psalmotoxin 1

Venom-derived peptide toxins can modify the gating characteristics of excitatory channels in neurons. How they bind and interfere with the flow of ions without directly blocking the ion permeation pathway remains elusive. Here we report the crystal structure of the trimeric chicken Acid-sensing ion channel 1 in complex with the highly selective gating modifier Psalmotoxin 1 at 3.0 Å resolution. The structure reveals the molecular interactions of three toxin molecules binding at the proton-sensitive acidic pockets of Acid-sensing ion channel 1 and electron density consistent with a cation trapped in the central vestibule above the ion pathway. A hydrophobic patch and a basic cluster are the key structural elements of Psalmotoxin 1 binding, locking two separate regulatory regions in their relative, desensitized-like arrangement. Our results provide a general concept for gating modifier toxin binding suggesting that both surface motifs are required to modify the gating characteristics of an ion channel.

Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist

The α(7) acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human α(7) AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native α(7). We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in α(7) AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in α(7) AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure-function relationships of α(7) AChRs.