Fig 2: Effects of intracellularly applied BMOE on hASIC1a activity and oligomerization.A: Currents were recorded in cut-open oocytes expressing either wild type ASIC1a (black bars n = 18) or ASIC1a-ΔCCt lacking cysteines in the C-terminus (grey bars, n = 17) before and after 10 s of intracellular perfusion with 2 mM BMOE (+BMOE). Bars represent mean ± SE. B: Anti-ASIC1a western from oocytes, either non-injected (n.i.), or expressing ASIC1a or ASIC1a-ΔCCt, untreated (left), or treated with 2 mM BMOE by internally perfusion (perf.) or intracellular injection (inj.). Numbers I, II, and IV designate the most prominent bands that are specific for ASIC1a, II and IV having apparent weight sizes that are, respectively, twice and four times that of I.

Fig 3: Light-activation of three acid-sensitive ion channels.(a) Principle of the two-component optogenetic (TCO) approach. Upon illumination the light-activated proton pump may moderately acidify the local extracellular medium and activate acid-sensitive ion channels, ASICs, via their proton-sensing domain. This results in a remote but large sodium influx that can be used for sustained cell depolarization at moderate light intensities. In Xenopus oocytes, a light-driven proton pump of Coccomyxa subellipsoidea (CsR) was used. (b–d) Macroscopic currents of CsRT46N coexpressed with rat ASIC1a (b), rat ASIC2a (c) or rat ASIC3 (d) in oocytes at a molar RNA ratio of 1:1 (for ASIC3 of 2:1). Cells were illuminated with 560 nm light at different holding voltages at 0.1 mM MOPS and pH 7.5 under constant perfusion. The small outward directed pump currents (CsR) triggers large inward sodium currents (ASIC). Inset is a zoom-in to the initial pump activity directly after starting to illuminate CsRT46N-ASIC1a with green light. Note that ASIC1a and ASIC3 show strong inactivation in sustained light, whereas ASIC2a shows moderate to no inactivation at all.

Fig 4: Validation of fractionation method with exogenously expressed proteins. (A). Representative immunofluorescence images of HEK cells expressing CFP-TGN-38 (top panels; arrows pointing at areas of cells with eGFP signal), eGFP (middle panels), and eGFP-ASIC1a (lower panels). Signal in green accounted for the fusion protein detected (left panels); PI (Propidium iodide) in red for nuclei (right panels); merge images (middle panel). Scale bar 10 um. (B) CFP-TGN-38-expressing cells were subjected to the fractionation protocol, and the lysates for each fraction were analyzed by Western blot experiments. As visualized in a representative membrane, the protein was enriched in the MO fraction (detected with an anti-GFP antibody), as expected, together with a marker for ER (using the anti-calnexin antibody). (C) eGFP-expressing cells were subjected to the fractionation protocol, and the lysates for each fraction were analyzed by Western blot experiments. As visualized in a representative membrane, the protein was enriched in the cytosolic, MO, and nuclear fractions, as expected. Each one of the fractions was confirmed to be enriched in proteins known to be abundant (GAPDH for Cyt; NaK for PM; CNX for MO (from the ER); and H3 for Nu). (D) eGFP-ASIC1a-expressing cells were subjected to the fractionation protocol, and the lysates for each fraction were analyzed by Western blot experiments. As visualized in a representative membrane, the protein was enriched in the PM and MO fractions. (E) Representative confocal microscopy images of HEK293 cells stained with an anti-ASIC1 antibody (and secondary Alexa-488, green), and with PI to visualize nuclei, showing a superficial section (top), a middle section (middle), and a merge of layers (bottom). Notice a predominant perinuclear staining. Scale bar 5 µm.

Fig 5: Expressions of ASIC subunits in rat cardiomyocytes.(a) Expressions of ASIC1, 2a and 3 proteins in rat cultured cardiomyocytes. Individual ASIC subunits peptides were added as negative controls. The blots were cropped from Supplementary Fig. S1a. The representative full-length blot for ASIC2a was shown in Supplementary Fig. S2. (b) Expressions of ASIC1, 2 and 3 transcripts in cultured rat ventricular myocytes. GAPDH transcript was used as control. M: marker; 1, 4: cardiomyocytes; 2: cortex; 3: negative control. (c) Double - labeling fluorescence of ASICs (green) and nucleus (blue, marker: Hoechst 33258) in cultured ratcardiomyocytes. NC: pretreatment with immunogenic peptide as negative control. Scale bars: 20 μm. All data were represented from at least three similar independent experiments.