Du C et al. (2025), Spider-derived peptide LCTx-F2 suppresses ASIC ...

XB-ART-61326

J Biol Chem 2025 Mar 10;3013:108286. doi: 10.1016/j.jbc.2025.108286.

Show Gene links Show Anatomy links

Spider-derived peptide LCTx-F2 suppresses ASIC channels by occupying the acidic pocket.

Du C , Yuan F , Zhang Z , He Z , Liu G , Hou W , Deng M , Liu C , Rong M .

???displayArticle.abstract???
Acid-sensing ion channels (ASICs) are proton-evoked sodium ion channels, highly distributed in the peripheral and central nervous system. ASICs are involved in pain perception, and ASIC3 channel is presumed as the target of promising analgesics. Peptide drugs have attracted the attention of pharmaceutical developers because of their advantages such as low toxic side effects and targeted specificity. Although numbers of chemicals acting on ASICs are emerging, there are limited reports on peptide inhibitor acting on ASIC3 channel. Here, we found that spider-derived peptide LCTx-F2 suppressed the activity of ASIC3 channel in a concentration-dependent manner. By performing peptide mutation and molecular docking, we revealed the molecular mechanism of LCTx-F2 inhibiting ASIC3 channel, in which β-hairpin of LCTx-F2 penetrated the acidic pocket of the channel. Similarly, LCTx-F2 also inhibited ASIC1a channel by occupying the acidic pocket, but N terminus of the peptide sticked into the region. The bond relationship between critical residues of LCTx-F2 and the channels was uncovered by molecular docking and dynamic simulation. Thus, our findings indicated the molecular mechanism by which LCTx-F2 acts on ASIC3 and ASIC1a channels and provided a novel template of analgesic drug targeting the channels.

???displayArticle.pubmedLink??? 39938802
???displayArticle.link??? J Biol Chem

Species referenced: Xenopus

???attribute.lit??? ???displayArticles.show???

	Figure 1. LCTx-F2 inhibited rASIC3 channel. A, the predicated structure of LCTx-F2. β-hairpin, Ω loop, and C terminus of LCTx-F2 were labeled. Some residues in LCTx-F2 are shown in stick representation. B and C, representative current traces (B) and the effect and concentration (C) of LCTx-F2 inhibiting rASIC3 channel evoked by proton. After 80 s perfusion of LCTx-F2 in pH 7.4 solution, the current of rASIC3 channel was elicited by proton in (B). The Hill equation was employed to fit the data in (C) (n = 4–5). ASIC, acid-sensing ion channel.
	Figure 2. The effect of mutants of LCTx-F2 on rASIC3 channel. A, the averaged inhibitory effect of 10.0 μM mutants of LCTx-F2 on rASIC3 channel. The current of rASIC3 channel was elicited by proton (pH 6.0 solution). The data were represented as the mean ± SD. ∗∗∗∗p < 0.0001, performed by one-way ANOVA followed by Dunnett’s post hoc test, significantly different of mutant group compared with WT group (n = 3). B, C, and E, the representative traces of mutants R7E (B), D14A (C), and K2E (E) inhibiting rASIC3 channel evoked by proton. D, the dose–response of some mutants of LCTx-F2 inhibiting rASIC3 channel as compared with WT of the receptor (n = 4). F, the dose–response of mutant K2E inhibiting rASIC3 channel as compared with WT of the receptor (n = 4). ASIC, acid-sensing ion channel.
	Figure 3. LCTx-F2 binding to rASIC3 channel. A, surface model of LCTx-F2 with the basic skeleton. β-hairpin and Ω loop of LCTx-F2 were labeled. Residues Lys2, Arg7, Asp10, Asp14, Arg15, and Arg20 were colored by blue. B and C, top view (B) and side view (C) of LCTx-F2 binding to rASIC3 channel. LCTx-F2 and rASIC3 channel were colored by cyan and green, respectively. D, close-up view of LCTx-F2 inserted into the acidic pocket of rASIC3 channel. LCTx-F2 were colored by cyan. Residue Arg183 in rASIC3 channel are shown in stick representation. Acidic pocket is shown by red circle, and helix α5 is colored raspberry. E, bond relationship of Asp10 (left) and Arg7 (right) in LCTx-F2 with rASIC3 channel. Asp10, Arg7 of LCTx-F2 and Gly117, Lys227, His109 of ASIC3 channel are shown in stick representation. Salt bridge, pi interaction, and hydrogen bond were colored by red, orange, and cyan, respectively. F, bond relationship of Glu36 in LCTx-F2 with rASIC3 channel. Salt bridge is colored red. G and H, saturation mutation energy of residues Glu36 (G) and Lys2 (H) in LCTx-F2 binding to rASIC3 channel. Mutation energy > +0.5 kcal/mol was considered as significantly different. I, the averaged inhibitory effect of 10.0 μM mutants of LCTx-F2 on rASIC3 channel. The current of rASIC3 channel was elicited by proton (pH 6.0 solution). The data were represented as the mean ± SD. ∗∗∗∗p < 0.0001, performed by one-way ANOVA followed by Dunnett’s post hoc test, significantly different of mutants group compared with WT group (n = 3). ASIC, acid-sensing ion channel.
	Figure 4. LCTx-F2 inhibiting rASIC1a channel. A, the sequence alignment of rASIC3 channel and rASIC1a channel at the acidic pocket is shown. The same residues both in rASIC3 channel and rASIC1a channel are indicated in black. B and C, representative current traces (B) and the averaged inhibitory effect (C) of 10.0 μM WT or mutants of LCTx-F2 inhibiting rASIC3 channel evoked by proton. After 80 s perfusion of LCTx-F2 in pH 7.4 solution, the current of rASIC3 channel was elicited by proton in (B). The data (C) were represented as the mean ± SD. ∗∗∗∗p < 0.0001 and ∗ p < 0.05 (R15E, 0.0195 and R20E, 0.0114), performed by one-way ANOVA followed by Dunnett’s post hoc test, significantly different of mutant group compared with WT group, respectively (n = 3). D, side view of snake toxin mambalgin1 binding to hASIC1a channel (Protein Data Bank ID: 7CFT). Toxin mambalgin1 and ASIC1a channel were colored as cyan and green, respectively. E, side view of toxins LCTx-F2 and PcTx1 binding to hASIC1a channel. LCTx-F2, PcTx1, and ASIC1a channel were colored as blue, cyan, and green, respectively. F, close-up view of LCTx-F2 inserted into the acidic pocket of ASIC1a channel. LCTx-F2 were colored by cyan. Residue Arg190 in ASIC1a channel is shown as sticks. Acidic pocket is shown by red circle, and helix α5 is colored raspberry. Asp14, Arg15, Arg20, Lys2, Arg7, Asp10 of LCTx-F2 and Arg175, Glu177, Glu235, Glu97, Arg190, Glu340, Phe352 of ASIC1a channel are shown as stick. G, bond relationship of some critical residues in LCTx-F2 with rASIC3 channel. Salt bridge, pi interaction, and hydrogen bond are colored by red, orange, and cyan, respectively. H, model of LCTx-F2 binding to ASIC1a channel. LCTx-F2 is shown in surface. Lys2 in LCTx-F2 is colored in blue, and some critical residues is colored by red. ASIC, acid-sensing ion channel.
	Figure 5. Dynamic simulation analysis of LCTx-F2 binding to ASIC3 channel or ASIC1a channel. A–C, three poses of rASIC3 channel and superimposed LCTx-F2′s conformation during dynamic simulation of 100 ns (10 frames, 10 ns/frame). For clarity, only a representative rASIC3 channel structure from a single frame is shown in green. D–F, salt bridges between residues of LCTx-F2 and rASIC3 channel during simulation of 100 ns, coming from the dynamic simulation (A–C). G, representative pose of hASIC1a channel and superimposed LCTx-F2′s conformation during dynamic simulation of 100 ns (10 frames, 10 ns/frame). For clarity, only a representative hASIC1a channel structure from a single frame is shown in yellow. H and I, salt bridges between residues of LCTx-F2 and hASIC1a channel during simulation of 100 ns, coming from the dynamic simulation (G). ASIC, acid-sensing ion channel.