A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603101113 ◽

2016 ◽

Vol 113 (43) ◽

pp. E6696-E6703 ◽

Cited By ~ 18

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.

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

The Journal of General Physiology ◽

10.1085/jgp.201110623 ◽

2011 ◽

Vol 138 (2) ◽

pp. 179-194 ◽

Cited By ~ 31

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.

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An arginine residue in the outer segment of hASIC1a TM1 affects both proton affinity and channel desensitization

The Journal of General Physiology ◽

10.1085/jgp.202012802 ◽

2021 ◽

Vol 153 (5) ◽

Author(s):

Zhuyuan Chen ◽

Georg Kuenze ◽

Jens Meiler ◽

Cecilia M. Canessa

Keyword(s):

Steady State ◽

Proton Affinity ◽

Outer Segment ◽

Pain Perception ◽

Transmembrane Domain ◽

High Energy ◽

Open Conformation ◽

Gating Mechanism ◽

Acid Sensing Ion Channels ◽

Channel Opening

Acid-sensing ion channels (ASICs) respond to changes in pH in the central and peripheral nervous systems and participate in synaptic plasticity and pain perception. Understanding the proton-mediated gating mechanism remains elusive despite the of their structures in various conformational states. We report here that R64, an arginine located in the outer segment of the first transmembrane domain of all three isoforms of mammalian ASICs, markedly impacts the apparent proton affinity of activation and the degree of desensitization from the open and preopen states. Rosetta calculations of free energy changes predict that substitutions of R64 in hASIC1a by aromatic residues destabilize the closed conformation while stabilizing the open conformation. Accordingly, F64 enhances the efficacy of proton-mediated gating of hASIC1a, which increases the apparent pH50 and facilitates channel opening when only one or two subunits are activated. F64 also lengthens the duration of opening events, thus keeping channels open for extended periods of time and diminishing low pH-induced desensitization. Our results indicate that activation of a proton sensor(s) with pH50 equal to or greater than pH 7.2–7.1 opens F64hASIC1a, whereas it induces steady-state desensitization in wildtype channels due to the high energy of activation imposed by R64, which prevents opening of the pore. Together, these findings suggest that activation of a high-affinity proton-sensor(s) and a common gating mechanism may mediate the processes of activation and steady-state desensitization of hASIC1a.

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Photolabelling the rat urotensin II/GPR14 receptor identifies a ligand-binding site in the fourth transmembrane domain

Biochemical Journal ◽

10.1042/bj20021566 ◽

2003 ◽

Vol 370 (3) ◽

pp. 829-838 ◽

Cited By ~ 35

Author(s):

Antony A. BOUCARD ◽

Simon S. SAUVÉ ◽

Gaétan GUILLEMETTE ◽

Emanuel ESCHER ◽

Richard LEDUC

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Transmembrane Domain ◽

Inositol Phosphate ◽

Receptor Complex ◽

Urotensin Ii ◽

Cognate Receptor ◽

Ligand Binding Sites ◽

Methionine Residues ◽

Mutant Receptors

A urotensin II (U-II) peptide analogue containing the photoreactive p-benzoyl-l-phenylalanine (Bz-Phe) in the sixth position was used to identify ligand-binding sites of the rat U-II receptor, also known as GPR14. [Bz-Phe6]U-II bound the receptor expressed in COS-7 cells with high affinity (IC50 0.7nM) and was as potent as U-II in the agonist-induced production of inositol phosphate. Photolabelling of the U-II receptor with 125I-[Bz-Phe6]U-II resulted in the specific formation of a glycosylated 125I-[Bz-Phe6]U-II—U-II receptor complex of 60kDa. Digestion of the 60kDa complex with endoproteinase Glu-C generated a fragment of 17kDa circumscribing the labelled fragment to residues 148—286. Digestion of the ligand—receptor complex with endoproteinase Arg-C produced a short peptide of 4kDa corresponding to fragments 125—148, 167—192 or 210—233. CNBr treatment of the endoproteinase-Glu-C and -Arg-C fragments yielded 2kDa fragments, defining the labelling site to methionine residues 184/185 of the fourth transmembrane domain. Photolabelling of two mutant receptors, M184L/M185L and M184A/M185A, led to a significant decrease in the overall yield of covalent labelling. Taken together, our results indicate that position 6 of U-II normally occupied by phenylalanine would interact with Met184 and/or Met185 of the fourth transmembrane domain of the U-II receptor. This information should be of significant value in the study of the interactions between U-II and its cognate receptor.

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Structural Comparisons of Ligand-gated Ion Channels in Open, Closed, and Desensitized States Identify a Novel Propofol-binding Site on Mammalian γ-Aminobutyric Acid Type A Receptors

Anesthesiology ◽

10.1097/aln.0000000000000588 ◽

2015 ◽

Vol 122 (4) ◽

pp. 787-794 ◽

Cited By ~ 25

Author(s):

Nicholas P. Franks

Keyword(s):

Ion Channel ◽

Chloride Channel ◽

Transmembrane Domain ◽

Type A ◽

Transmembrane Domains ◽

Aminobutyric Acid ◽

State Dependent ◽

Novel Approach ◽

Channel Opening ◽

Acid Type

Abstract Background: Most anesthetics, particularly intravenous agents such as propofol and etomidate, enhance the actions of the neurotransmitter γ-aminobutyric acid (GABA) at the GABA type A receptor. However, there is no agreement as where anesthetics bind to the receptor. A novel approach would be to identify regions on the receptor that are state-dependent, which would account for the ability of anesthetics to affect channel opening by binding differentially to the open and closed states. Methods: The open and closed structures of the GABA type A receptor homologues Gloeobacter ligand–gated ion channel and glutamate-gated chloride channel were compared, and regions in the channels that move on channel opening and closing were identified. Docking calculations were performed to investigate possible binding of propofol to the GABA type A β3 homomer in this region. Results: A comparison between the open and closed states of the Gloeobacter ligand–gated ion channel and glutamate-gated chloride channel channels identified a region at the top of transmembrane domains 2 and 3 that shows maximum movement when the channels transition between the open and closed states. Docking of propofol into the GABA type A β3 homomer identified two putative binding cavities in this same region, one with a high affinity and one with a lower affinity. Both cavities were adjacent to a histidine residue that has been photolabeled by a propofol analog, and both sites would be disrupted on channel closing. Conclusions: These calculations support the conclusion of a recent photolabeling study that propofol acts at a site at the interface between the extracellular and transmembrane domains, close to the top of transmembrane domain 2.

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Ligand recognition and gating mechanism through three ligand-binding sites of human TRPM2 channel

eLife ◽

10.7554/elife.50175 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Yihe Huang ◽

Becca Roth ◽

Wei Lü ◽

Juan Du

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Neurological Disorders ◽

Transmembrane Domain ◽

Structural Rearrangements ◽

Channel Activation ◽

Physiological Processes ◽

Gating Mechanism ◽

Trpm2 Channel ◽

Ligand Binding Sites

TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2’s activation by Ca2+ and ADP ribose (ADPR), an NAD+-metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca2+ in the transmembrane domain. The 8-Br-cADPR—an antagonist of cADPR—binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology.

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