Acid-sensing ion channels emerged over 600 Mya and are conserved throughout the deuterostomes

Lynagh, Timothy; Mikhaleva, Yana; Colding, Janne M; Glover, Joel C.; Pless, Stephan A.

doi:10.1073/pnas.1806614115

Cited by 59 publications

(105 citation statements)

References 54 publications

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“…This constitutes an important advantage over existing methodologies. Specifically, we anticipate that the approach will overcome some of the drawbacks associated with conventional genetic engineering in eukaryotic cells (nonspecific incorporation, premature termination, dependence on ribosomal promiscuity 36,43 ) and semi-synthetic approaches that require protein refolding 7 . It will thus increase the number and type of functionalities that can be incorporated into proteins that prove amenable to tPTS.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Toadfish ASIC2 channels containing A18′ were expressed in oocyte membranes but showed no proton-gated currents (Coric et al, 2005), reflecting results of the E18′A mutation in mASIC1a, which abolished channel activity (Lynagh et al, 2017a). Sea urchin and acorn worm ASIC subunits possessing a Q18′ residue and from a distinct subclade of ASICs showed low Na + /K + selectivity (Lynagh et al, 2018), reflecting results of E18′Q mutations in mASIC1a and rASIC2a.…”

Section: Discussionmentioning

confidence: 99%

Determinants of ion selectivity in ASIC1a- and ASIC2a-containing acid-sensing ion channels

Lynagh

Flood

Boiteux

et al. 2020

Journal of General Physiology

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Trimeric acid-sensing ion channels (ASICs) contribute to neuronal signaling by converting extracellular acidification into excitatory sodium currents. Previous work with homomeric ASIC1a implicates conserved leucine (L7′) and consecutive glycine-alanine-serine (GAS belt) residues near the middle, and conserved negatively charged (E18′) residues at the bottom of the pore in ion permeation and/or selectivity. However, a conserved mechanism of ion selectivity throughout the ASIC family has not been established. We therefore explored the molecular determinants of ion selectivity in heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels using site-directed mutagenesis, electrophysiology, and molecular dynamics free energy simulations. Similar to ASIC1a, E18′ residues create an energetic preference for sodium ions at the lower end of the pore in ASIC2a-containing channels. However, and in contrast to ASIC1a homomers, ion permeation through ASIC2a-containing channels is not determined by L7′ side chains in the upper part of the channel. This may be, in part, due to ASIC2a-specific negatively charged residues (E59 and E62) that lower the energy of ions in the upper pore, thus making the GAS belt more important for selectivity. This is confirmed by experiments showing that the L7′A mutation has no effect in ASIC2a, in contrast to ASIC1a, where it eliminated selectivity. ASIC2a triple mutants eliminating both L7′ and upper charges did not lead to large changes in selectivity, suggesting a different role for L7′ in ASIC2a compared with ASIC1a channels. In contrast, we observed measurable changes in ion selectivity in ASIC2a-containing channels with GAS belt mutations. Our results suggest that ion conduction and selectivity in the upper part of the ASIC pore may differ between subtypes, whereas the essential role of E18′ in ion selectivity is conserved. Furthermore, we demonstrate that heteromeric channels containing mutations in only one of two ASIC subtypes provide a means of functionally testing mutations that render homomeric channels nonfunctional.

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“…Despite the potential pathophysiological relevance of the ASIC-BigDyn interaction (10) is not likely to be the primary proton sensor of ASICs (37,38), side chains in and around the acidic pocket have been shown to modulate proton sensitivity (30,37). A direct binding of BigDyn to this region is therefore expected to alter proton sensing.…”

Section: Mechanism Of Action Of Bigdyn On Asic1amentioning

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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Acid-sensing ion channels emerged over 600 Mya and are conserved throughout the deuterostomes

Cited by 59 publications

References 54 publications

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

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

Determinants of ion selectivity in ASIC1a- and ASIC2a-containing acid-sensing ion channels

Mechanism and site of action of big dynorphin on ASIC1a

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