Fig. 6: E⁶EEE⁹ at distal N-terminus of ASIC1a are critical for intracellular auto-inhibition.

a Rosetta model of closed state ASIC1a suggests that negatively charged glutamate residues (E⁶EEE⁹) are juxtaposed to positively charged lysines (K⁴⁶⁸, K⁴⁷¹, and K⁴⁷⁴) in the CT death motif, forming electrostatic interactions (upper). The distance between beta carbons of E⁷ and K⁴⁶⁸ is ~4.7 Å. With E → A substitutions at E⁶EEE⁹ (lower), ASIC1a-NT moves apart from the lysines at CT death motif. (b) The E/A mutant exhibited lower FRET than WT ASIC1a at pH 7.4. CFP and YFP were tagged at NT and CT, respectively. The YFP/CFP emission ratios were acquired under the same conditions using identical settings. n = 12–14, * p < 0.05 vs. WT by unpaired t test. c, d Expression of E/A mutant in CHO cells resulted in more cell death than WT ASIC1a at pH 7.4. Representative images of PI-stained cells (c) and quantification of PI-positive CHO cells (d). n > 200 transfected neurons (green) counted for each. Scale bar, 20 µm. * p < 0.05 vs. WT by unpaired t test. e Co-IP experiments of transfected CHO cells showing that E/A mutant exhibited increased association with NSF and RIPK1 at pH 7.4. f, g Summary data for ASIC1a-NSF (f) and ASIC1a-RIPK1 (g) association as determined in (e). Data are normalized to NSF/ASIC1a and RIPK1/ASIC1a of WT group at pH 7.4, respectively. n = 3, * p < 0.05 vs. corresponding pH 7.4, ^## p < 0.01 vs. WT, by ANOVA. h LDH assay showing that the E → A substituted NT_1–20 peptide (NT_1–20^E/A, 10 μM) failed to exert neuroprotection against acidotoxicity of cultured cortical neurons, contrasting to Nec-1 (20 μM) and peptide NT_1–20 (10 μM). n = 4, *** p < 0.001 vs. corresponding pH 7.4, ^### p < 0.001, ns, no statistical significance vs. NT_1–20 at pH 6.0, by ANOVA. i, j Representative images of brain sections (i) and summary data (j) showing that pre-administration of peptide NT_1–20, but not peptide NT_1–20^E/A, by microinfusion reduced infarct volumes in mouse MCAO model. KO is ASIC1a KO. n = 6, *** p < 0.001 vs. WT with Ctrl peptide, by ANOVA.