scholarly journals Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening

2011 ◽  
Vol 138 (2) ◽  
pp. 179-194 ◽  
Author(s):  
Iehab Talukder ◽  
Lonnie P. Wollmuth
Keyword(s):  
Nmda Receptor ◽  
Ligand Binding ◽  
Ion Channel ◽  
Nmda Receptors ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Functional Feature ◽  
Channel Pore ◽  
Activation Gating ◽  
Pore Opening

The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel–forming domain, composed of M1–M4 segments, are connected by three linkers: S1–M1, M3–S2, and S2–M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3–S2 and S2–M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process.

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

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Laura Fedele ◽  
Joseph Newcombe ◽  
Maya Topf ◽  
Alasdair Gibb ◽  
Robert J. Harvey ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Ligand Binding ◽  
Ion Channel ◽  
Missense Mutations ◽  
Receptor Ligand ◽  
Ion Channel Properties ◽  
Channel Properties

scholarly journals Cryo-EM Structure of OSCA1.2 from Oryza sativa: Mechanical basis of hyperosmolality-gating in Plants

2018 ◽  
Author(s):  
Koustav Maity ◽  
John Heumann ◽  
Aaron P McGrath ◽  
Noah J Kopcho ◽  
Po-Kai Hsu ◽  
...  
Keyword(s):  
Ion Channel ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Turgor Pressure ◽  
Environmental Water ◽  
Structural Basis ◽  
Molecular Architecture ◽  
Calcium Dependent ◽  
Lateral Tension ◽  
And Function

Sensing and responding to environmental water deficiencies is essential for the growth, development and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolarity in Arabidopsis. Here, we report the cryo-EM structure and function of an ion channel from rice (Oryza stativa; OsOSCA1.2), showing how it mediates hyperosmolality sensing and ion permeability. The structure reveals a dimer, the molecular architecture of each subunit consists of eleven transmembrane helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The transmembrane domain is structurally related to the TMEM16 family of calcium dependent ion channels and scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms parallel to the plasma membrane and well positioned to sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the transmembrane domain to open the channel and HDX mass spectrometry experimentally confirmed the conformational dynamics of these coupled domains. The structure provides a framework to understand the structural basis of hyperosmolality sensing in crop plants, extending our knowledge of the anoctamin superfamily important for plants and fungi as well as structural mechanisms that can translate membrane stress to ion transporter regulation.

scholarly journals Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

2016 ◽  
Vol 113 (43) ◽  
pp. E6696-E6703 ◽  
Author(s):  
Mieke Nys ◽  
Eveline Wijckmans ◽  
Ana Farinha ◽  
Özge Yoluk ◽  
Magnus Andersson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Ion Channel ◽  
Binding Site ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Channel Pore ◽  
X Ray ◽  
Electrophysiological Recordings ◽  
Channel Opening ◽  
Dynamics Simulations

Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8–β9 loop in the extracellular ligand-binding domain. The β8–β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8–β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.

scholarly journals A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

2015 ◽  
Vol 146 (4) ◽  
pp. 323-340 ◽  
Author(s):  
Nicolaus Schmandt ◽  
Phanindra Velisetty ◽  
Sreevatsa V. Chalamalasetti ◽  
Richard A. Stein ◽  
Ross Bonner ◽  
...  
Keyword(s):  
Ion Channel ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Primary Amines ◽  
Molecular Architecture ◽  
X Ray Crystallography ◽  
Gated Channel ◽  
Activation Mechanisms ◽  
Double Electron Electron Resonance

Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

scholarly journals Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

2020 ◽  
Author(s):  
Fei Jin ◽  
Minxuan Sun ◽  
Takashi Fujii ◽  
Yurika Yamada ◽  
Jin Wang ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Channel Activity ◽  
State Structure ◽  
Channel Pore ◽  
Closed State ◽  
Channel Inactivation ◽  
Pore Opening ◽  
Functional Analyses ◽  
Pore Domain ◽  
Cytoplasmic Side

ABSTRACTMgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear.Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

scholarly journals Energetic Coupling of the Ligand Binding Domain to Pore Opening in NMDA Receptors

2014 ◽  
Vol 106 (2) ◽  
pp. 29a ◽  
Author(s):  
Rashek Kazi ◽  
Jian Dai ◽  
Melissa Daniel ◽  
Huan-Xiang Zhou ◽  
Lonnie P. Wollmuth
Keyword(s):  
Ligand Binding ◽  
Nmda Receptors ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Pore Opening

scholarly journals Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow

2015 ◽  
Vol 112 (47) ◽  
pp. 14705-14710 ◽  
Author(s):  
Kim Dore ◽  
Jonathan Aow ◽  
Roberto Malinow
Keyword(s):  
Nmda Receptor ◽  
Ligand Binding ◽  
Ion Channel ◽  
Cytoplasmic Domain ◽  
Agonist Binding ◽  
Cytoplasmic Domains ◽  
Ion Flow ◽  
Brain Physiology ◽  
Antibody Targeting ◽  
Neuronal Compartments

The NMDA receptor (R) plays important roles in brain physiology and pathology as an ion channel. Here we examine the ion flow-independent coupling of agonist to the NMDAR cytoplasmic domain (cd). We measure FRET between fluorescently tagged cytoplasmic domains of GluN1 subunits of NMDARs expressed in neurons. Different neuronal compartments display varying levels of FRET, consistent with different NMDARcd conformations. Agonist binding drives a rapid and transient ion flow-independent reduction in FRET between GluN1 subunits within individual NMDARs. Intracellular infusion of an antibody targeting the GluN1 cytoplasmic domain blocks agonist-driven FRET changes in the absence of ion flow, supporting agonist-driven movement of the NMDARcd. These studies indicate that extracellular ligand binding to the NMDAR can transmit conformational information into the cell in the absence of ion flow.

scholarly journals Dynamical model of the CLC-2 ion channel exhibits a two-step gating mechanism

2017 ◽  
Author(s):  
Keri A. McKiernan ◽  
Anna K. Koster ◽  
Merritt Maduke ◽  
Vijay S. Pande
Keyword(s):  
High Resolution ◽  
Ion Channel ◽  
Chloride Channel ◽  
Conformational Dynamics ◽  
Selectivity Filter ◽  
Ion Conduction ◽  
Channel Pore ◽  
Conduction Pathway ◽  
Additional State ◽  
The Brain

AbstractThis work reports a dynamical Markov state model of CLC-2 “fast” (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low (∼1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population (∼1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.Author summaryIn the brain, the roles and mechanisms of sodium-, potassium-, and calcium-selective ion channels are well established. In contrast, chloride-selective channels have been studied much less and are not sufficiently understood, despite known associations of chloride-channel defects with brain disorders. The most broadly expressed voltage-activated chloride channel in the brain is CLC-2 (one of 9 human CLC homologs). In this work, we use simulations to model the conformational dynamics of the CLC-2 chloride ion channel selectivity filter (SF), which is the part of the protein that controls whether the channel is in an ion-conducting or non-conducting state. Our analysis identifies four primary conformational states and a specific progression through these states. Our results are consistent with structural and functional data in the literature and provide a high-resolution model for guiding further studies of CLC-2. These results will inform our understanding of how CLC-2 governs electrical activity and ion homeostasis in the brain.

scholarly journals NMDA Receptor Opening and Closing—Transitions of a Molecular Machine Revealed by Molecular Dynamics

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 546 ◽  
Author(s):  
Jiří Černý ◽  
Paulína Božíková ◽  
Aleš Balík ◽  
Sérgio M. Marques ◽  
Ladislav Vyklický
Keyword(s):  
Molecular Dynamics ◽  
Nmda Receptor ◽  
Ion Channel ◽  
Structural Transition ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Molecular Machine ◽  
Large Rearrangement ◽  
Extracellular Domains ◽  
Opening And Closing

We report the first complete description of the molecular mechanisms behind the transition of the N-methyl-d-aspartate (NMDA) receptor from the state where the transmembrane domain (TMD) and the ion channel are in the open configuration to the relaxed unliganded state where the channel is closed. Using an aggregate of nearly 1 µs of unbiased all-atom implicit membrane and solvent molecular dynamics (MD) simulations we identified distinct structural states of the NMDA receptor and revealed functionally important residues (GluN1/Glu522, GluN1/Arg695, and GluN2B/Asp786). The role of the “clamshell” motion of the ligand binding domain (LBD) lobes in the structural transition is supplemented by the observed structural similarity at the level of protein domains during the structural transition, combined with the overall large rearrangement necessary for the opening and closing of the receptor. The activated and open states of the receptor are structurally similar to the liganded crystal structure, while in the unliganded receptor the extracellular domains perform rearrangements leading to a clockwise rotation of up to 45 degrees around the longitudinal axis of the receptor, which closes the ion channel. The ligand-induced rotation of extracellular domains transferred by LBD–TMD linkers to the membrane-anchored ion channel is responsible for the opening and closing of the transmembrane ion channel, revealing the properties of NMDA receptor as a finely tuned molecular machine.

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