High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Braun, Nina; Friis, Søren; Ihling, Christian; Sinz, Andrea; Andersen, Jacob; Pless, Stephan A.

doi:10.1101/2020.11.24.392498

Cited by 5 publications

(11 citation statements)

References 90 publications

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“…These experiments indicate that PcTx1 interacts with F350L* and introduces conformational changes, despite the lack of functional consequences at these concentrations. This is in line with earlier results that suggested PcTx1 to still bind to F352L hASIC1a, as pre-incubation with PcTx1 prevented the modulatory effects of BigDyn and PcTx1 could still be crosslinked with the mutant channel (Braun, Friis et al 2021). Similarly, experiments from ASIC1a/2a concatemers of different subunit stoichiometries suggested that PcTx1 also binds to ASIC1a-2a interfaces (Joeres, Augustinowski et al 2016), despite the fact that ASIC2a subunits contain a leucine at the position corresponding to 350 in mASIC1a.…”

Section: The F350l Mutation Destabilizes a Pctx1-induced Conformation...supporting

confidence: 90%

Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

Heusser

Borg

Colding

et al. 2022

eLife

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Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.

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Section: The F350l Mutation Destabilizes a Pctx1-induced Conformation...supporting

confidence: 90%

Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

Heusser

Borg

Colding

et al. 2022

eLife

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View full text Add to dashboard Cite

show abstract

“…These experiments indicate that PcTx1 interacts with F350L* and introduces conformational changes, despite the lack of functional consequences at these concentrations. This is in line with earlier results that suggested for PcTx1 to still bind to F352L hASIC1a, as pre-incubation with PcTx1 prevented the modulatory effects of BigDyn and PcTx1 could still be crosslinked with the mutant channel (Braun, Friis et al 2021). Similarly, experiments from ASIC1a/2a concatemers of different subunit stoichiometries suggested that PcTx1 also binds to ASIC1a-2a interfaces (Joeres, Augustinowski et al 2016), despite the fact that ASIC2a subunits contain a Leu at the position corresponding to 350 in mASIC1a.…”

Section: The F350l Mutation Destabilizes a Pctx1-induced Conformational Statessupporting

confidence: 91%

Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

Borg

Heusser

Colding

et al. 2021

Preprint

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Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that contribute to fast synaptic transmission. Pharmacological inhibition of ASIC1a has been shown to reduce neurotoxicity and infarct volumes during stroke. The cysteine knot toxin Psalmotoxin-1 (PcTx1) is one of the most potent and selective inhibitors of ASIC1a. PcTx1 binds at the subunit interface, but both the stoichiometric requirements and the dynamics of the conformational consequences of the ion channel-peptide interaction remain unknown. Here, we use a combination of electrophysiology, voltage-clamp fluorometry and subunit concatenation to decipher the mechanism of PcTx1 inhibition. We observe a long-lived PcTx1-induced conformational change in the ASIC1a extracellular domain that is destabilized by the F350L mutation at the PcTx1 binding site. Concatemeric channel constructs show that two WT ASIC1a subunits are sufficient for WT-like current inhibition, while the presence of a single mutated subunit is enough to destabilize the PcTx1-induced conformation. Our results therefore demonstrate a divergence between the functional effects of PcTx1 on the pore and its conformational consequences in the extracellular domain. It further highlights how engineering of ion channels enables precise control over individual subunits for pharmacological and conformational assessment to determine the mechanism of ion channel-ligand interactions.

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“…One 12-well dish contained ECSs with resting pH (7.4), and another dish contained ECSs with activating pH (pH 6). ECSs were applied using a liquid stacking approach as described elsewhere ( Braun et al, 2021 Preprint ). The pipette tips were loaded with 25 µl pH 7.4 solution followed by 15 μl pH 6.0 solution.…”

Section: Methodsmentioning

confidence: 99%

The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs

Sheikh

Wulf

Friis

et al. 2021

Journal of General Physiology

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The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid–based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.

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High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Cited by 5 publications

References 90 publications

Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs

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