Figure 4. A peptide representing the proximal C-terminal region of ASIC1a induces RIP1 phosphorylation and neuronal death.
(A) Relative positions and amino acid sequences of four peptides representing different regions of the mouse ASIC1a (mASIC1a) C-terminus. (B) TAT-tagged CP-1, but not CP-2, CP-3, or CP-4, peptide (10 μM, 24 hr) induced neuronal death at pH 7.4 (CTB assay, n=3, ***p<0.001; NS, no statistical significance, vs control [CTRL, the TAT peptide alone]). (C, D) CP-1, but not CP-2, CP-3, or CP-4, enhanced RIP1 phosphorylation (C) and the effect was blocked by Nec-1 (20 µM) (D). (E) Nec-1 (20 μM) partially rescued CP-1-induced neuronal death (CTB assay, n=3, ***p<0.001 vs CTRL; ###p<0.001 vs CP-1 alone).
Figure 4—figure supplement 1. Design and characterization of ASIC1a-derived peptides.
(A) Alignment of ASIC C-termini of different isoforms from various species. Starting positions are indicated in parentheses. Locations of the four mouse ASIC1a C-terminal peptides are shown by the boxes. c, chicken; f, toad fish; h, human; m, mouse; r, rat; s, shark; z, zebrafish. (B) CP-1 failed to inhibit acid-induced neuronal death (CTB assay, n=3, ***p<0.001 vs pH 7.4; ##p<0.001 vs pH 6.0+CP-1). (C) CP-1, but not CP-3, caused the death of CHO cells in the absence of ASIC1a (CTB assay, n=3, ***p<0.001; NS, no statistical significance, vs CTRL, the TAT peptide alone).