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. 2020 Sep 30;37(2):145–165. doi: 10.1007/s12264-020-00581-9

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Screening extracellular HA-tagged hASIC1a for surface staining. A Candidate sites for epitope tag insertion in the extracellular loop of hASIC1a. Based on the high-resolution structure, two exposed flexible sites were chosen for epitope insertion, the first between Phe¹⁴⁷ and Lys¹⁴⁸ (upper panel) and the second between Asp²⁹⁸ and Leu²⁹⁹ (lower panel). B Representative images of surface staining by HA (red) and total hASIC1a expression by GFP (green) in Asic1a^−/− mouse cortical neurons that expressed different HA-tagged GFP-hASIC1a as indicated at left. mCherry was co-transfected to indicate neuronal morphology (magenta). Scale bars: upper panel, 20 μm; lower panel, 10 μm. C Quantification of surface hASIC1a labeling in neurons based on HA/GFP fluorescence intensity ratio. Data are presented as the mean ± SEM. The ratios for ¹⁴⁷2HA¹⁴⁸ (0.775 ± 0.018, n = 8 cells) and ²⁹⁸HA²⁹⁹ (0.747 ± 0.005, n = 6 cells) are higher than that for ¹⁴⁷HA¹⁴⁸ (0.307 ± 0.022, n = 9 cells). ****P < 0.0001, no significant difference between ¹⁴⁷2HA¹⁴⁸ and ²⁹⁸HA²⁹⁹, one-way ANOVA multiple comparison. D, E Representative blots (D) and quantification (E) of surface (biotinylated, S) and total (T) hASIC1a in CHO cells that expressed WT and the three HA-tagged hASIC1a as indicated. GAPDH was used as a cytoplasmic protein control. The normalized surface/total ratios are presented as the mean ± SEM: ¹⁴⁷HA¹⁴⁸, 0.89 ± 0.10; ¹⁴⁷2HA¹⁴⁸, 0.99 ± 0.10; ²⁹⁸HA²⁹⁹, 0.90 ± 0.05. No significant difference between WT and any HA-tagged hASIC1a, one-way ANOVA multiple comparison, n = 3 experiments. F Representative traces of acid (pH 6.0)-activated currents recorded from CHO cells transfected with WT or HA-tagged hASIC1a as indicated. Cells were held at −60 mV; baseline pH was 7.4. G Quantification of peak current density. Data are presented as the mean ± SEM. The density of I_6.0 (pA/pF) for ¹⁴⁷HA¹⁴⁸ (10.92 ± 3.80, n = 9 cells), ¹⁴⁷2HA¹⁴⁸ (15.46 ± 3.57, n = 19 cells), and ²⁹⁸HA²⁹⁹ (48.78 ± 6.54, n = 18 cells), is lower than that of WT hASIC1a (259.5 ± 26.61, n = 29 cells). ***P < 0.001, one-way ANOVA multiple comparison. H pH dose-response curves for WT and HA-tagged hASIC1a (normalized to current elicited by pH 4.0 solution). To ensure that the channels were not desensitized before stimulation, the baseline pH was 9.0. pH₅₀ (data are presented as the mean ± SEM): WT, 6.13 ± 0.05 (n = 13 cells); ¹⁴⁷HA¹⁴⁸, 5.78 ± 0.06 (n = 13 cells); ¹⁴⁷2HA¹⁴⁸, 5.13 ± 0.04 (n = 15 cells); and ²⁹⁸HA²⁹⁹, 5.67 ± 0.08 (n = 12 cells). I, J GFP-hASIC1a-²⁹⁸HA²⁹⁹ displays the same localization as WT hASIC1a (represented by mCherry-hASIC1a). GFP-hASIC1a-²⁹⁸HA²⁹⁹ and mCherry-hASIC1a were co-transfected into Asic1a^−/− neurons. I Representative images of GFP and mCherry, showing excellent co-localization with a Pearson’s coefficient (r) of 0.917. Scale bars: left panel, 50 μm; right panel, 10 μm. J Intensity plots for the line profile of GFP and mCherry along the dashed lines shown in (I).