Polyunsaturated fatty acid structure determines the strength of potentiation of Acid-sensing ion channels

Mapping Intimacies ◽

10.1101/2021.06.25.449979 ◽

2021 ◽

Author(s):

Robert C Klipp ◽

John R Bankston

Keyword(s):

Ion Channels ◽

Ph Dependence ◽

Lipid Binding ◽

Structural Basis ◽

Side Chain ◽

Head Group ◽

Arginine Side Chain ◽

Acid Sensing Ion Channels ◽

Acid Structure ◽

Positively Charged

Acid-sensing ion channels (ASICs) are thought to be endogenous sensors of acidic pain in inflammatory pathways. It has previously been demonstrated that arachidonic acid (AA), a pain and inflammation promoting molecule, potentiates ASICs. However, a mechanistic understanding of how AA regulates ASICs is lacking. Furthermore, little is known regarding modulation by other polyunsaturated fatty acids (PUFAs). Here we show that PUFAs stabilize the open state of the channel by shifting the pH dependence of activation to more alkaline values, increasing max conductance, and slowing channel desensitization. We examine the effects of 35 PUFAs/PUFA derivatives and show that ASICs can be more strongly potentiated by these lipids than was originally seen for AA. In fact, arachidonoyl glycine (AG) can act as a ligand and activate the channel in the absence of acidic pH. We find that the strength of potentiation is critically dependent upon a negatively charged PUFA head group as well as both the length and the number of doubles bonds in the acyl tail. PUFA-induced shifts in the pH dependence of activation could be eliminated upon mutation of a highly conserved, positively charged arginine in the outer segment of TM1 (R64). Combined our results suggest a hypothesis whereby an electrostatic interaction between the charged PUFA head group and the positively charged arginine side chain potentiates ASIC currents by stabilizing the open state of the channel. This work uncovers a novel putative lipid binding site on ASICs and provides the structural basis for future development of compounds targeting ASICs.

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Structure of a Talaromyces pinophilus GH62 arabinofuranosidase in complex with AraDNJ at 1.25 Å resolution

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18000250 ◽

2018 ◽

Vol 74 (8) ◽

pp. 490-495 ◽

Cited By ~ 3

Author(s):

Olga V. Moroz ◽

Lukasz F. Sobala ◽

Elena Blagova ◽

Travis Coyle ◽

Wei Peng ◽

...

Keyword(s):

Plant Biomass ◽

Cellulosic Biomass ◽

Structural Basis ◽

Side Chain ◽

Polymeric Substrates ◽

Hydrolysis Of ◽

Insight Into ◽

Positively Charged ◽

Talaromyces Pinophilus ◽

Resolution Structure

The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a K d of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.

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A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition

Journal of Experimental Medicine ◽

10.1084/jem.20051777 ◽

2006 ◽

Vol 203 (3) ◽

pp. 661-673 ◽

Cited By ~ 90

Author(s):

Lars Kjer-Nielsen ◽

Natalie A. Borg ◽

Daniel G. Pellicci ◽

Travis Beddoe ◽

Lyudmila Kostenko ◽

...

Keyword(s):

Hot Spot ◽

Cell Receptor ◽

Nkt Cells ◽

Structural Basis ◽

Head Group ◽

Antigen Specificity ◽

Nkt Cell ◽

Plasticity Mechanism ◽

Positively Charged ◽

Selection Of

Little is known regarding the basis for selection of the semi-invariant αβ T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d–glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3β composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR α- and semi-invariant β-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved “hot spot” that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3β loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR–CD1d–glycolipid interaction in which the invariant CDR3α loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant αβ TCR and the unique antigen specificity of NKT cells.

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Insights into the Mechanism of Pore Opening of Acid-sensing Ion Channel 1A

Journal of Biological Chemistry ◽

10.1074/jbc.m110.202366 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16297-16307 ◽

Cited By ~ 27

Author(s):

Lindsey A. Tolino ◽

Sora Okumura ◽

Ossama B. Kashlan ◽

Marcelo D. Carattino

Keyword(s):

Disulfide Bonds ◽

Underlying Mechanism ◽

Side Chain ◽

Extracellular Acidification ◽

Extracellular Region ◽

Acid Sensing Ion Channels ◽

Outer Vestibule ◽

Pore Opening ◽

Double Mutant Cycle ◽

Positively Charged

Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains.

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Acid-sensing ion channels (ASICs) are differentially modulated by anions dependent on their subunit composition

AJP Cell Physiology ◽

10.1152/ajpcell.00216.2012 ◽

2013 ◽

Vol 304 (1) ◽

pp. C89-C101 ◽

Cited By ~ 13

Author(s):

Nobuyoshi Kusama ◽

Mamta Gautam ◽

Anne Marie S. Harding ◽

Peter M. Snyder ◽

Christopher J. Benson

Keyword(s):

Ion Channels ◽

Ph Dependence ◽

Extracellular Fluid ◽

Dorsal Root Ganglion Neurons ◽

Subunit Composition ◽

Fluid Composition ◽

Whole Cell Patch Clamp ◽

Acid Sensing Ion Channels ◽

Ganglion Neurons ◽

Kinetics Of

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. ASIC1a channels possess intersubunit Cl−-binding sites in the extracellular domain, which are highly conserved between ASIC subunits. We previously found that anions modulate ASIC1a gating via these sites. Here we investigated the effect of anion substitution on native ASICs in rat sensory neurons and heterologously expressed ASIC2a and ASIC3 channels by whole cell patch clamp. Similar to ASIC1a, anions modulated the kinetics of desensitization of other ASIC channels. However, unlike ASIC1a, anions also modulated the pH dependence of activation. Moreover, the order of efficacy of different anions to modulate ASIC2a and -3 was very different from that of ASIC1a. More surprising, mutations of conserved residues that form an intersubunit Cl−-binding site in ASIC1a only partially abrogated the effects of anion modulation of ASIC2a and had no effect on anion modulation of ASIC3. The effects of anions on native ASICs in rat dorsal root ganglion neurons mimicked those in heterologously expressed ASIC1a/3 heteromeric channels. Our data show that anions modulate a variety of ASIC properties and are dependent on the subunit composition, and the mechanism of modulation for ASIC2a and -3 is distinct from that of ASIC1a. We speculate that modulation of ASIC gating by Cl− is a novel mechanism to sense shifts in extracellular fluid composition.

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Structural Basis for Interactions of the Phytophthora sojae RxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-12-0184-r ◽

2013 ◽

Vol 26 (3) ◽

pp. 330-344 ◽

Cited By ~ 58

Author(s):

Furong Sun ◽

Shiv D. Kale ◽

Hugo F. Azurmendi ◽

Dan Li ◽

Brett M. Tyler ◽

...

Keyword(s):

Host Cell ◽

Lipid Binding ◽

Phytophthora Sojae ◽

Effector Proteins ◽

Cell Entry ◽

Structural Basis ◽

Minor Role ◽

Head Group ◽

Host Cell Entry ◽

Phosphatidylinositol 3

Oomycetes such as Phytophthora sojae employ effector proteins that enter plant cells to facilitate infection. Entry of some effector proteins is mediated by RxLR motifs in the effectors and phosphoinositides (PIP) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effectors involved in PIP recognition. We have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that Avh5 is helical in nature, with a long N-terminal disordered region. NMR titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate, directly identified the ligand-binding residues. A C-terminal lysine-rich helical region (helix 2) was the principal lipid-binding site, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Mutations in the RFLR motif or in the basic region both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. Based on these findings, we propose a model where Avh5 interacts with PtdIns(3)P through its C terminus, and by binding of the RFLR motif, which promotes host cell entry.

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Mutation of a conserved glutamine residue does not abolish desensitization of acid-sensing ion channel 1

The Journal of General Physiology ◽

10.1085/jgp.202012855 ◽

2021 ◽

Vol 153 (8) ◽

Author(s):

Matthew L. Rook ◽

Megan Miaro ◽

Tyler Couch ◽

Dana L. Kneisley ◽

Maria Musgaard ◽

...

Keyword(s):

Ion Channels ◽

Single Channel ◽

Side Chain ◽

Accelerated Molecular Dynamics ◽

Acid Sensing Ion Channels ◽

The Common ◽

Bond Network ◽

Dynamics Simulations ◽

Gated Ion Channels ◽

Ligand Gated Ion Channels

Desensitization is a common feature of ligand-gated ion channels, although the molecular cause varies widely between channel types. Mutations that greatly reduce or nearly abolish desensitization have been described for many ligand-gated ion channels, including glutamate, GABA, glycine, and nicotinic receptors, but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine (Q276G) in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and the single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1, the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes, essentially like wild-type, when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, the Q276G mutation also reduces desensitization, but not to the extent reported previously. Interestingly, the kinetic consequences of Q276G depend on the human variant used. In the common G212 variant, Q276G slows desensitization, while in the rare D212 variant desensitization accelerates. Our data reveal that while the Q/G mutation does not abolish or substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.

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Mutation of a conserved Gln residue does not abolish desensitization of acid-sensing ion channel 1

10.1101/2020.12.19.423606 ◽

2020 ◽

Author(s):

Matthew L Rook ◽

Megan Miaro ◽

Tyler Couch ◽

Dana L Kneisley ◽

Maria Musgaard ◽

...

Keyword(s):

Ion Channels ◽

Single Channel ◽

Side Chain ◽

Accelerated Molecular Dynamics ◽

Ph Values ◽

Acid Sensing Ion Channels ◽

Bond Network ◽

Dynamics Simulations ◽

Gated Ion Channels ◽

Ligand Gated Ion Channels

AbstractDesensitization is a common feature of ligand-gated ion channels although the molecular cause varies widely between channel types. Mutations that substantially reduce or abolish desensitization have been described for many ligand-gated ion channels including glutamate, GABA, glycine and nicotinic receptors but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1 the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes essentially like wild type when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, Q276G does not substantially reduce desensitization but surprisingly slows entry to and exit from the desensitized state, thus requiring longer agonist applications to reach equilibrium. Our data reveal that while the Q/G mutation does not substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.

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