A selectivity filter at the intracellular end of the acid-sensing ion channel pore

Lynagh, Timothy; Flood, E. A.; Boiteux, Céline; Wulf, Matthias; Komnatnyy, Vitaly V.; Colding, Janne M; Allen, Toby W.; Pless, Stephan A.

doi:10.7554/elife.24630

Cited by 56 publications

(150 citation statements)

References 84 publications

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“…Figure 2A illustrates one such patch where the variance for 50 111 consecutive sweeps was calculated and plotted as a function of the current amplitude ( Figure 112 2B). The NSFA indicated a peak Popen of 0.86 ± 0.02 (n = 5, Figure 2C) with an estimated single 113 channel conductance of 10 ± 1 pS, consistent with previously published conductance data 19 115 (Lynagh et al, 2017;Zhang and Canessa, 2002). Importantly, to our knowledge this represents open probability without substantially altering proton potency (Figures 1 and 2, Sup Fig 1).…”

supporting

confidence: 89%

β11-12 linker isomerization governs Acid-sensing ion channel desensitization and recovery

Rook¹,

Williamson

Lueck

et al. 2019

Preprint

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1 Acid-sensing ion channels (ASICs) are neuronal sodium-selective channels activated by reductions 2 in extracellular pH. Structures of the three presumptive functional states, high-pH resting, low-3 pH desensitized, and toxin-stabilized open, have all been solved for chicken ASIC1. These 4 structures, along with prior functional data, suggest that the isomerization or flipping of the β11-5 12 linker in the extracellular, ligand-binding domain is an integral component of the 6 desensitization process.To test this, we combined fast perfusion electrophysiology, molecular 7 dynamics simulations and state-dependent non-canonical amino acid cross-linking. We find that 8 both desensitization and recovery can be accelerated by orders of magnitude by mutating resides 9 in this linker or the surrounding region. Furthermore, desensitization can be suppressed by 10 trapping the linker in the resting state, indicating that isomerization of the β11-12 linker is not 11 merely a consequence of, but a necessity for the desensitization process in ASICs. 12

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supporting

confidence: 89%

β11-12 linker isomerization governs Acid-sensing ion channel desensitization and recovery

Rook¹,

Williamson

Lueck

et al. 2019

Preprint

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“…This is particularly true for complex proteins expressed in eukaryotic cells. In fact, many groups have repeatedly observed yields of 10% or less with ncAA incorporation into transporters 36 , ion channels [37][38][39] , and G protein-coupled receptors 40,41 . Although the generally low yields observed with tPTS likely restrict the approach to applications that do not require large amounts of protein, at least some of the above limitations can be addressed by engineering more promiscuous and efficient split inteins [10][11][12]42 or by adding affinity tags to promote split intein interactions 18 .…”

Section: Discussionmentioning

confidence: 99%

“…cRNAs were injected into Xenopus laevis oocytes (prepared as previously described 38 ) and incubated at 18 °C in OR-3 solution (50 % Leibovitz's medium, 1 mM L-Glutamine, 250 mg/L Gentamycin, 15 mM HEPES, pH 7.6) for up to 7 days. For injection of synthetic peptides, lyophilized peptides were dissolved in Milli-Q H2O to a concentration of 750 µM and 18 nL of solubilized peptide was injected into cRNA pre-injected oocytes with the Nanoliter 2010 micromanipulator (World Precision Instruments).…”

Section: Expression In Xenopus Laevis Oocytesmentioning

confidence: 99%

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Khoo

Galleano

Gasparri

et al. 2020

Preprint

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AbstractManipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, NaV1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.

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“…Oocytes were surgically removed from adult female Xenopus laevis and prepared as previously described (56). Oocytes were injected with 0.1 to 10 ng cRNA of mouse ASIC1a (mASIC1a) or hASIC1a mRNA into the cytosol (volumes between 9 and 50 nL).…”

Section: Electrophysiological Recordings and Data Analysismentioning

confidence: 99%

Mechanism and site of action of big dynorphin on ASIC1a

Borg

Braun

Heusser

et al. 2019

Preprint

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Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to synaptic plasticity, as well as initiation of pain and neuronal death following ischemic stroke.As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acidmediated photocrosslinking. We demonstrate that BigDyn binding induces ASIC1a conformational changes that are different from those induced by protonation and likely represent a distinct closed state. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.Note: This manuscript has not been peer-reviewed 3

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A selectivity filter at the intracellular end of the acid-sensing ion channel pore

Cited by 56 publications

References 84 publications

β11-12 linker isomerization governs Acid-sensing ion channel desensitization and recovery

β11-12 linker isomerization governs Acid-sensing ion channel desensitization and recovery

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Mechanism and site of action of big dynorphin on ASIC1a

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