Journal: PLoS Biology

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

doi: 10.1371/journal.pbio.3001321

Figure Lengend Snippet: (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.

Article Snippet: To assess modulation by PcTx1 (Alomone Labs, Israel, >95% purity), cells were exposed to 2 control measurements of activation with pH 5.6 after conditioning at pH 7.4 (interval 3.75 minutes), followed by pH 5.6 activation after incubation with 100 nM PcTx1 at varying pH (pH 7.4 to 7.0) for 2 minutes (total interval between stimuli 7 minutes), as well as 2 further controls to assess recovery from modulation.

Techniques: Incubation, Variant Assay, Activation Assay, MANN-WHITNEY, Standard Deviation

Journal: PLoS Biology

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

doi: 10.1371/journal.pbio.3001321

Figure Lengend Snippet: (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower ECD (right insets). Positions that crosslinked to biotin-PcTx1 are colored red, F352 is marked in orange, and positions that did not crosslink are colored green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 minutes to form covalent hASIC1a–PcTx1 complexes, which are purified via a carboxyl-terminal 1D4-tag on hASIC1a and visualized via western blotting. (C) Western blot of purified hASIC1a WT, UT cells, and variants carrying AzF in the ECD detected using the specified AB. Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356, and 357 in the acidic pocket (colored red in A, left inset), but not at positions 177, 236, 239, 343, or 351 (colored green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower ECD (right insets in A), WT, or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 colored orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data are representative of 3 individual experiments; see – Figs for original blots and crosslinking attempts with Bpa. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 12 and 13). AB, antibodies; ASIC1a, acid-sensing ion channel 1a; AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; cASIC1, chicken acid-sensing ion channel 1; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; PcTx1, psalmotoxin 1; PDB, Protein Data Bank; UT, untransfected; WT, wild type.

Article Snippet: To assess modulation by PcTx1 (Alomone Labs, Israel, >95% purity), cells were exposed to 2 control measurements of activation with pH 5.6 after conditioning at pH 7.4 (interval 3.75 minutes), followed by pH 5.6 activation after incubation with 100 nM PcTx1 at varying pH (pH 7.4 to 7.0) for 2 minutes (total interval between stimuli 7 minutes), as well as 2 further controls to assess recovery from modulation.

Techniques: Expressing, Incubation, Purification, Western Blot, Mutagenesis