Journal: bioRxiv

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

doi: 10.1101/2020.11.24.392498

Figure Lengend Snippet: (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and six ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 min (pink bars) before activation at pH 5.6 (grey bars, 5 sec) and the currents were normalized to the average of two control currents after conditioning at pH 7.6 (black bars; control traces shown in Figure S11). (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 min (blue bars) before activation at pH 5.6 (grey bars, 5 sec) and the current was normalized to the average of the four preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± S.D, dashed line indicates 100%, values in Table S5 and S6. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann Whitney test (B) or one-way ANOVA with Tukey’s multiple comparisons test (D). Coloured and black bars in (A) and (C) not to scale.

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

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

Journal: bioRxiv

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

doi: 10.1101/2020.11.24.392498

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 extracellular domain (right insets). Positions that crosslinked to biotin-PcTx1 are coloured red, F352 is marked in orange and positions that did not crosslink are coloured 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 min to form covalent hASIC1a-PcTx1 complexes, which are purified via a C-terminal 1D4-tag on hASIC1a and visualized via Western blotting. (C) Western blot of purified hASIC1a WT, untransfected cells (UT) and variants carrying AzF in the extracellular domain detected using the specified antibodies (AB). Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356 and 357 in the acidic pocket (coloured red in A, left inset), but not at positions 177, 236, 239, 343 or 351 (coloured green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower extracellular domain (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 coloured 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 is representative of three individual experiments, see Figures S12-15 for original blots and crosslinking attempts with Bpa.

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

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