Short RFamide, CFamide and FCamide peptides as
novel positive modulators of ASIC 3 with similar
potentiating effects but different reversibility

Maurizio Toft, Maëva Meynier, Hélène Lubrano Di Scampamorte, Cédric Vallée, Miguel Salinas, Peijun Zhang, Jessica Tacco, Anne-Sophie Gay, Emmanuel Bourinet, Eric Lingueglia, et al.

► To cite this version:

Maurizio Toft, Maëva Meynier, Hélène Lubrano Di Scampamorte, Cédric Vallée, Miguel Salinas, et al. Short RFamide, CFamide and FCamide peptides as novel positive modulators of ASIC 3 with similar potentiating effects but different reversibility. Neuropharmacology, 2025, 287, pp.110813. 10.1016/j.neuropharm.2025.110813. hal-05443894v2

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# Short RFamide, CFamide and FCamide peptides as novel positive modulators of ASIC3 with similar potentiating effects but different reversibility

Maurizio Toft<sup>a,1 ID</sup>, Maëva Meynier<sup>a,1 ID</sup>, Hélène Lubrano Di Scampamorte<sup>a ID</sup>, Cédric Vallée<sup>b</sup>, Miguel Salinas<sup>a ID</sup>, Peijun Zhang<sup>b,c ID</sup>, Jessica Tacco<sup>a</sup>, Anne-Sophie Gay<sup>a</sup>, Emmanuel Bourinet<sup>d</sup>, Eric Lingueglia<sup>a,2</sup>, Emmanuel Deval<sup>a,*2 ID</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

ASIC

Amidated hexapeptides

Pharmacology

computational modeling

Structure-function

## ABSTRACT

Acid-sensing ion channels (ASICs) are members of the DEG/ENaC family that includes the only known peptide-gated ion channels. While ASICs are gated by protons, they are also sensitive to peptides and are modulated by the molluscan FMRFamide and other mammalian neuropeptides ending by the RFamide motif. We identified a set of synthetic short amidated hexapeptides, which not only end by the RFamide motif but also by CFamide and FCamide, as potent positive modulators of ASIC3 acid-induced activity. We focused on two of them, a RFamide peptide (FRCRFamide) and a CFamide peptide (FRCRCFamide), demonstrating that they have similar specificity for and effects on ASIC3. The potentiating effects of the two peptides are due to a strong slow-down of desensitization, leading to an increase in the amount of current induced by acid pH (≤pH6.6), with apparent affinities ranging from 1 to 5 µM. Surprisingly, the washout kinetic of FRCRFamide peptide was much slower than those of FRCRCFamide and other known RFamide peptides, suggesting potential differences in their mechanisms of action. Computational modeling and structure-function analysis reveal interactions of both peptides with the non-proton binding site of ASIC3 as already reported before for other RFamide peptides, but our data also suggest possible additional effects of FRCRFamide involving directly or indirectly the proton binding domain. These findings expand our understanding of ASICs' modulation by peptides, identifying novel short modulators of ASIC3, including peptides with new CFamide and FCamide ending motifs, and showing differences between these peptides using their washout kinetic as a new parameter.

## 1. Introduction

Several members of the degenerin/epithelial sodium channel family of ion channels (DEG/ENaC) have the interesting property of being directly modulated by peptides (for review, see (Grunder et al., 2023)). Some invertebrate members of this ion channel family (i.e., FaNaCs (Lingueglia et al., 1995) from snails, HyNaCs from hydra (Golubovic et al., 2007) and MGIC from a marine annelid (Schmidt et al., 2018)) even represent the only known peptide-gated ion channels, as they are

directly opened by RFamide (FaNaCs and HyNaCs) or Wamide (MGIC) peptides. Mammalian Acid-Sensing Ion Channels (ASICs) also belong to the DEG/ENaC family. They are gated by extracellular proton and display both transient and sustained depolarizing activities triggered by extracellular acidification (for reviews see (Deval et al., 2010; Wemmie et al., 2013)), and also alkalization in the case of human ASIC3 (Delaunay et al., 2012). Although they are not peptide-gated ion channels, ASICs are also sensitive to peptides, as they are positively modulated by the canonical molluscan FMRFamide and other endogenous

<footnote><sup>a</sup> Université Côte d'Azur, CNRS UMR7275, Inserm U1323, IPMC, LabEx ICST, FHU InovPain, Valbonne 06560, France</footnote>

<footnote><sup>b</sup> Electron Bio-Imaging Centre (eBIC), Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, United Kingdom</footnote>

<footnote><sup>c</sup> Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom</footnote>

<footnote><sup>d</sup> IGF, Montpellier University, CNRS, INSERM, Montpellier 34000, France</footnote>

<footnote>* Corresponding author. should be addressed to
E-mail address: deval@ipmc.cnrs.fr (E. Deval).</footnote>

<footnote n="1">¹ Co-first authors.</footnote>

<footnote n="2">² Co-last authors.</footnote>
RFamide-related neuropeptides, which mainly target ASIC3, by dynor-
phin A and big dynorphin targeting ASIC1a, and by YFMRFamide that
modulates ASIC1b and ASIC3 (Askwith et al., 2000; Catarsi et al., 2001;
Deval et al., 2003; Lingueglia et al., 2006; Reimers et al., 2017; Reiners
et al., 2018; Vyvers et al., 2018). While the existence of endogenous
peptide(s) able to directly gate ASICs remains an open question, no
endogenous peptide activator has been found so far despite extensive
search.

RFamide peptides are effectors of ASICs, mainly ASIC3 but also ASIC1a, which are positively modulated by FMRFamide, neuropeptide FF (NPFF), neuropeptide SF (NPSF), YFMRFamide and RPRFamide, with EC50 ranging from 2 to 50 µM (Askwith et al., 2000; Catarsi et al., 2001; Deval et al., 2003; Lingueglia et al., 2006; Reimers et al., 2017; Reiners et al., 2018; Vyvers et al., 2018). The RFamide motif at the end of these peptides appears very important for their effects, i.e. adding this motif to Met-enkephalin that has no effect on ASIC3 is sufficient to produce an effect comparable to that of FMRFamide (Lingueglia et al., 2006), although RFamide alone remains much less efficient than FMRFamide, NPFF or NPSF (Deval et al., 2003). More recently, a RPRFamide peptide isolated from Conus textile venom has been reported to target ASIC3, enhancing its activity as well as acid-induced muscle pain (Reimers et al., 2017). This cono-RFamide peptide has been proposed to stabilize the open conformation of ASIC3 through binding to its non-proton binding domain (Reiners et al., 2018), which could be a common mechanism for all RFamide peptides.

About thirty years ago, seven positively-charged cyclic amidated
hexapeptides were described as novel blockers of the cardiac sodium-
calcium exchange (NCX), with IC50 ranging from 10 µM to more than
250 µM (Khananshvili et al., 1995). Part of these synthetic peptides
display a C-terminal RFamide motif, while others display a C-terminal
CFamide or FCamide motif, raising the question of whether they can
modulate ASICs, particularly ASIC3. Here, we report that these peptides,
including two non-RFamide ones with a C-terminal CFamide or FCamide
motif, are indeed novel positive modulators of ASIC3 and we focus on
two of them: (i) the FRCRCFamide, which is not a RFamide peptide and
has the best NCX inhibitory activity (Khananshvili et al., 1995) and (ii)
the FRCRFamide, which is a RFamide and has a 15-fold lower inhibi-
tory effect on NCX (Khananshvili et al., 1995). Both peptides display a
similar potentiating effect on ASIC3, with EC50 in the micromolar range
and interaction sites within the non-proton binding site of the channel
initially identified for the synthetic compound GMQ (2-guanidine-4--
methylquinazoline) (Yu et al., 2010). This is in line with what has
already been proposed for other RFamide peptides (Reiners et al., 2018).
However, the washout kinetic for the FRCRFamide peptide was much
slower than those of FRCRCFamide and other RFamide peptides, sug-
gesting a more complex mechanism of action also possibly involving the
proton binding domain.

## 2. Methods

2.1. *Plasmid constructions and mutagenesis*

The coding sequences of rat ASIC1a, ASIC3 and human ASIC3a and their related mutants were subcloned into pIRES2-eGFP vector (Addgene). Rat ASIC1b, ASIC2a and ASIC2b were subcloned into pCI vector (Promega). Mutants of rat ASIC3 were obtained by recombinant PCR strategies as previously described (Salinas et al., 2009).

## 2.2. Cell culture and transfection

HEK293 cell were grown in DMEM medium (Lonza™ Bio-
Whittaker™) supplemented with 10 % of heat-inactivated fetal bovine
serum (Biomest™) and 1 % of antibiotics (penicillin + streptomycin,
Lonza™ BioWhittaker™). One day after plating, cells were transfected
with either pIRES2-ratASIC1a-EGFP, pCI-ratASIC1b + pIRES2-eGFP,

pCI-ratASIC2a + pIRES2-eGFP, pCI-ratASIC2b + pIRES2-eGFP, pIRES2-ratASIC3-EGFP, pIRES2-humanASIC3a-EGFP vector using the JetPEI reagent according to the supplier's protocol (Polyplus transfection SA, Illkirch, France). Fluorescent cells were used for patch-clamp recordings 2-4 days following transfection.

2.3. Primary cultures of DRG neurons

Primary cultures of DRG neurons were performed on adult male C57Bl6J wild-type mice (Janvier) and ASIC3 knockout mice (Wultsch et al., 2008) breed in animal facilities of IPMC, which agreement has been approved by the local ethical committee and the French government (agreement n°F06-152-5). Lumbar dorsal root ganglia (DRG L1 to L6) were rapidly dissected out from euthanized adult male C57Bl6J (Janvier Lab, at least 8 weeks of age) and ASIC3<sup>-/-</sup> mice, and placed in cold Ca<sup>2+</sup>-free/Mg<sup>2+</sup>-free HBSS medium (Corning®) supplemented with 10 mM glucose, and 5 mM HEPES (pH7.5 with NaOH). After carefully removing the roots, DRGs were enzymatically dissociated at 37 °C for 2 × 20min in the HBSS solution supplemented with calcium (CaCl<sub>2</sub> 5 mM) and containing type II collagenase (2 g/L, Gibco, France) and dispase (Gibco, France). DRGs were then mechanically triturated and washed in a complete neurobasal A medium (NBA, Gibco® Invitrogen™ supplemented with 2 % B27, 2 mM L-glutamine and 1 % antibiotics: penicillin + streptomycin, Lonza™ BioWhittaker™), before being plated in 35-mm petri dishes. Neurons were then kept in culture at 37 °C for one week with 1/3 of the culture medium (complete NBA supplemented with 10 ng/ml NT3, 2 ng/ml GDNF, 10 ng/ml BDNF, 100 ng/ml NGF and 100 nM retinoic acid) renewed every 2 days, and were used for patch-clamp experiments at least 1 day after plating.

## 2.4. Patch-clamp experiments

Membrane currents were recorded using patch-clamp in the whole cell configuration (voltage-clamp mode). Patch pipettes (3–7MΩ) were made from borosilicate glass tubes (Hilgenberg, Germany) using a P30 puller (Sutter instruments, USA), before being filled with an intracellular solution containing either 135 mM KCl, 2.5 mM ATP-Na₂, 2.1 mM CaCl₂, 5 mM EGTA, 2 mM MgCl₂, 10 mM HEPES (pH7.3 with KOH) for DRG neurons, or 135 mM KCl, 5 mM NaCl, 5 mM EGTA, 2 mM MgCl₂, 10 mM HEPES, (pH7.3 with KOH) for HEK293 cells. Cells were bathed into an extracellular solution made of 145 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂ and 10 mM HEPES (or MES depending on the final pH of the solution), and this medium was supplemented with 10 mM glucose for DRG neurons. Extracellular pH was then adjusted to its final value using NMDG, including HEPES-containing solutions for 7.4≤pH ≤ 6.6 and MES-containing solutions for 6.6<pH ≤ 5.0. During patch-clamp recordings, the cell under investigation was continuously superfused with extracellular solutions using a homemade eight-outlet system digitally controlled by solenoid valves (Warner Instruments), allowing rapid changes of the immediate cell environment from control to test solutions. Electrophysiological signals generated by patch-clamped cells were amplified and low-pass filtered at 2 kHz using an Axopatch 200B amplifier (Molecular Devices, UK), digitized with a 1440 A-D/D-A converter (Molecular Devices, UK), sampled at 20 kHz and stored on a computer using Clampex software (V10.7, Molecular Devices). Off-line analyses were then made using Clampfit software (V10.7, Molecular Devices). In experiments conducted on DRG neurons, the capacitance was 14.7 ± 1.3 pF (n = 15) and 18.6 ± 2.0 pF (n = 6) for WT and ASIC3 KO neurons, respectively; p = 0.12, unpaired t-test). Neurons exhibiting an ASIC current in response to a pH5.5 extracellular acidification were distinguished from those that did not according to the following criteria: transient peak current followed by a desensitization phase and possibly a sustained current. The minimum peak current recorded in ASIC neurons at pH5.5 was 300 pA.
## 2.5. Chemicals

All the peptides used in this study were synthesized by the SB Peptides company (SB Peptides, France) in either their cyclic or linear form, and mass spectrometry data were provided for each peptide, indicating 2m/z differences between cyclic and linear forms. They were all received lyophilized, reconstituted at 1 mM stock solutions (stored at -20 °C) in either water or external patch-clamp medium, before being freshly prepared at their final concentration (1 nM–100 µM) for patch-clamp experiments. In order to avoid peptides adsorption to the perfusion tubing, BSA 0.05 % was added to the resting pH7.4 extracellular patch-clamp solution. When two peptides have been applied on the same cells and in order to avoid any potential cumulative effect, the first peptide was washed until return to a baseline current before applying the second one. In addition, the order of application of FRCRCFamide and FRC<sup>CC</sup>RFamide peptides was randomized on each cell in order to avoid any bias. Finally, chromatography and spectrometry analyses were performed (see section 2.6) on some of the final samples used for patch-clamp experiments to confirm the presence/absence of a disulfide bridge (Supplementary Fig. 4D-E), showing that if the cyclic peptides actually had S-S bridge (Supplementary Fig. 4D), a small but significant part of the linear peptides could spontaneously reform one (Supplementary Fig. 4E).

## 2.6. Peptide analysis by NanoHPLC-HRMS

NanoHPLC-HRMS analysis was performed using a nanoRSLC system (ultimate 3000, Thermo Fisher Scientific) coupled to an Easy Exploris 480 (Thermo Fisher Scientific). Peptide separation was carried out using the Easy-Spray RSLC ultra high-performance LC system. 5 µL of peptide solution at 2,5 µM diluted into 0.1 % formic acid (FA) was injected and concentrated on a µ-Precolumn Cartridge Acclaim PepMap 100 C18 (i.d. 5 mm, 5 mm, 100 Å, Thermo Fisher Scientific) at a flow rate of 10 µL/min and using solvent containing H₂O / ACN / TFA 98 % / 2 % / 0.1 % (TFA: trifluoroacetic acid). Next, peptide separation was performed on a 75 mm i.d. x 150 mm (2 µm, 100 Å) Easy-SprayPepMap RSLC C18 column (Thermo Fisher Scientific) at a flow rate of 300 nL/min. Solvent systems were: (A) 100 % H₂O, 0.1 % FA, (B) 100 % ACN (acetonitrile), 0.1 % FA. The following gradient was used t = 0 min 2 % B; t = 3 min, 2 %B; t = 15 min, 90 % B; t = 18 min, 90 % B (temperature was regulated at 40 °C). MS spectra were acquired at a resolution of 120 000 (200 m/z) in a mass range of 200–1000 m/z with an AGC target 3e6 value of and a maximum injection time of 25 ms. Raw data were processed using FreeStyleTM 1.3 software.

## 2.7. Computational methods

The 3D structures of the homotrimeric ASIC3 was predicted using AlphaFold3 (Abramson et al., 2024) (https://alphafoldserver.com/) using the *Rattus norvegicus* ASIC3 sequence from Uniprot (entry O35240). Residues 495 to 533 (C-ter) were removed from the structure due to low pLDDT scores (less than 50) and lack of secondary structure. Based on comparison with several known ASIC1a structures, the predicted ASIC3 model is likely to be in the resting/closed state as its transmembrane domains yield the lowest RMSD value with a closed channel (PDB entries 7CFS & 7CFT (Sun et al., 2020), RMSD = 5.33 Å); and its acidic pockets yield the lowest RMSD with a channel in resting state at high pH (PDB entry 6VTL (Yoder and Gouaux, 2020), RMSD = 1.34 Å). The resulting rASIC3 structure was then embedded in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayer membrane, solvated in water with the TIP3P model (Pekka and Nilsson, 2001), and neutralized with 0.15 M of NaCl using the CHARMM-GUI Membrane Builder input generator (Lee et al., 2016). The system was subsequently minimized using the steepest decent algorithm with position restraints on the heavy atoms of the protein and the lipids, then was equilibrated

by 6 equilibration steps following the recommendation from CHARMM-GUI. A short Molecular Dynamics simulation was performed for 10 ns on the equilibrated system and a cluster analysis was performed using the 'gmx cluster' command from GROMACS to extract the most representative conformation of the embedded rASIC3 (referred to as the 'post-MD' structure). The 3D structures of both peptides (FRCRCFamide and FRC<sup>CC</sup>RFamide) were generated using the 'Build Structure' tool from ChimeraX version 1 (Meng et al., 2023). Docking of both peptides was then performed on the extracellular domain (search box size of 100 Å × 100 Å × 100 Å) of both the AlphaFold structure and the post-MD structure from the cluster analysis using the AutoDock Vina extension (version 1.1.2) of Chimera version 1.17.3 (Pettersen et al., 2004; Trott and Olson, 2010). Further docking of both peptides (search box size of 25 Å × 25 Å × 25 Å) were performed on the two sites with the most occupancies (determined from the results of the extracellular docking) referred to as the 'proton site' and the 'non-proton site'. As rASIC3 is an homotrimer, 3 dockings per site and per peptide were performed. The docking poses with the best score (i.e., with the lowest empirical estimation of the binding free energy, see (Gaillard, 2018) for more details about Vina's scoring function) for both peptides were used to generate new systems containing the peptide in one of the two sites of rASIC3. Only one pose from each docking was kept (leading to a total of three different poses for the proton site and a total of three different poses for the non-proton site as well, due to ASIC3 being homotrimeric). The ASIC3-peptide complex was then embedded in a POPC membrane using CHARMM-GUI. The CT2 terminal group patching was used for the peptides to make sure they have the amide functional group at their C-ter. Then, 3 independent MD replicates of each system with either peptide in the different docking sites were produced for 30 ns, for a total of 2160 ns of simulation. The initial docking poses were using as the starting coordinates of the peptide for the MD simulations. Finally, after discarding the first 5 ns of the simulations, the trajectories of each run were further analyzed using ProLIF (Protein-Ligand Interaction Fingerprints) (Bouysset and Fiorucci, 2021) to identify interactions between the peptide and the channel and the 'gmx energy' command to quantify the interaction energies for all residues interacting with the peptides for more than 50 % of the simulation in at least one of the replicates. MD production runs were performed in the NPT ensemble using the V-rescale thermostat and the C-rescale barostat to maintain the temperature and the pressure at 310.15 K and 1 bar, respectively. The leap-frog integrator was used with a timestep of 2 fs, and all covalent hydrogen bonds were constraint with the LINCS algorithm (Hess et al., 1998). The cutoff for the short range non-bonded interactions was 1.2 nm, and the long-range electrostatic interactions were evaluated using the Particle Mesh Ewald (PME) methods (Essmann et al., 1955). All simulations were carried with GROMACS version 2024.1 with CUDA GPU support, and the CHARMM36m forcefield (Huang and MacKerell, 2013; BEKKER et al., 1993).

## 2.8. Statistical analysis

Data are presented as mean ± SEM and statistical analyses were performed using GraphPad Prism software. Differences between sets of data were considered significant when *p*-values were less or equal to 0.05. Significant differences between datasets were calculated from either parametric or non-parametric tests followed by adequate *post-hoc* tests (see figure legends).

# 3. Results

## 3.1. The cyclic FRCRCFamide peptide is a selective potentiator of ASIC3 current

Among the cyclic hexapeptides initially synthesized as inhibitors of NCX activity, FRCRCFamide was reported as the one with the best af-
finity (IC₅₀ ~10 µM, see (Khananshvili et al., 1995)). Although this peptide does not contain the final RFamide motif, we found it to be a strong and quite selective potentiator of ASIC3 activity evoked at pH5.5 in HEK293 transfected cells (Fig. 1). This effect was characterized by a typical slow-down of the current desensitization when the peptide was applied at 10 µM in the resting/conditioning pH7.4, leading to an increase of its pH5.5-evoked sustained component (see the sustained current amplitude measured 5 s after the current onset, Fig. 1A), with no significant effect on the transient peak current (Fig. 1A–B). Conversely, this effect was not observed when the peptide was applied in the acid test solution, including at pH5.5 (Fig. 1A–B) and the less acidic pH values of 7.0 and 6.6 associated to slower desensitization (Supplementary Fig. 1). Finally, co-applying the peptide both in the pH7.4 and pH5.5 extracellular solutions did not induce any supplemental effect compared to application at pH7.4 only (Fig. 1A–B). The potentiating effect of FRCRCFamide on pH5.5-evoked ASIC3 sustained activity was highly variable (between 1.9- and 41.8-fold increase,

Supplementary Fig. 2A), and it was negatively correlated to the basal pH5.5-sustained current amplitude evoked in control condition, i. e. without peptide. Indeed, the strongest effects of FRCRCFamide (≥20-fold increase) were observed when the absolute value of the basal pH5.5-evoked sustained current amplitudes were the smallest (-0.5 to -6.0 pA/pF), and FRCRCFamide effect potency decreased as the absolute value of the basal pH5.5-evoked sustained current amplitudes increased (Supplementary Fig. 2A).

The effect of FRCRCFamide was also observed when the peptide concentration was reduced to 1 µM (Fig. 1C–D), with still a significant potentiation of the pH-evoked ASIC3 sustained activity while its transient peak amplitude remained unaffected (Supplementary Fig. 2B). The potentiation was strongest when ASIC3 sustained currents were generated by relatively more acidic pH (≤pH6.6), i.e., outside the ASIC3 window current (Deval et al., 2008; Marra et al., 2016; Yagi et al., 2006).

The FRCRCFamide peptide was next tested on different ASIC homomeric channels expressed in HEK293 cells (Fig. 1E–F), including

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** FRCRCFamide acts on the close state of ASIC3 to potentiate its low acid pH-induced sustained activity. **A–B**, Typical effects of FRCRCFa peptide applied extracellularly at 10 µM on pH5.5-evoked rat ASIC3 currents recorded at –50mV in HEK293 transfected cells (A). FRCRCFa peptide (10 µM) was either applied into the pH7.4 resting solution (conditioning, cond), the test pH5.5 solution (pulse) or both (cond + pulse, n = 8–25; ****p < 0.0001, mixed effect analysis followed by a Dunnett multiple comparison test) (B). **C–D**, Effect of FRCRCFa applied at 1 µM in the pH7.4 resting solution on ASIC3 current evoked by different pH (n = 4, *p* > 0.05, **p** < 0.001 and ***p** < 0.0001, Two-way ANOVA followed by a Sidak's multiple comparison test). **E**, Typical effects of FRCRCFa (10 µM) on homomeric rASIC1a, rASIC1b, rASIC2a, rASIC2b, rASIC3 and hASIC3a currents. **F**, Fold increase factors of the peak and sustained currents (Ipeptide/Icontrol, I/ctrl) generated by FRCRCFa (10 µM) on the different ASICs shown in E (n = 4–6, *p* < 0.05, paired t-test). As no significant pH5.5-evoked current was recorded for ASIC2b with or without peptide, the fold increase factor is not represented for this channel (n = 4).</figcaption></figure>
ASIC1a, ASIC1b, ASIC2a and ASIC2b, in addition to ASIC3. Except for ASIC3 (both rat and human clones), none of the other ASIC homomeric channels had their acid-evoked currents potentiated when FRCRCFamide (10 µM) was applied extracellularly in the resting/conditioning pH7.4, showing a relatively good selectivity toward ASIC3 compared to other ASICs, at least at the tested concentration of 10 µM (Fig. 1E–F).

### 3.2. The cyclic FCRRCFamide, FRCCCRFAmide and FCRCRFamide peptides also potentiate ASIC3 current

We next compared the effects on ASIC3 of the FRCRCFamide peptide with those of other cyclic hexapeptides ending by either the Famide or RFamide motif, i.e. FCRRCFamide and FRCCCRFAmide, but also FCRCRFAmide (Fig. 2). Those three peptides were found to have a similar effect on ASIC3 activity, i.e., a drastic potentiation of its pH5.5-evoked sustained current when applied at 10 µM in the resting pH7.4, with no significant effect on the peak current amplitude (Fig. 2A–B). Because these effects were associated to a slowdown of desensitization, a precise analysis of this current phase was performed with and without

peptides (Fig. 2C), showing no significant difference between the effects of the four peptides. Overall, the RFamide, CFamide and FCamide cyclic hexapeptides displayed similar potentiating effects on ASIC3, and we next focused our investigations on two of them: FRCRCFamide and FRCCCRFAmide.

### 3.3. FRCRCFamide and FRCCCRFAmide have different washout kinetics

The on/off kinetics of the effects of both FRCRCFamide and FRCCCRFAmide on ASIC3 (Fig. 3) were analyzed by first varying the application time of each peptide (10 µM in pH7.4 resting solution, Fig. 3A). We found that the onset of FRCCCRFAmide effect was very similar to that of FRCRCFamide, with a half maximum effect reached in 1–2 s and a maximal effect in ~20 s (Fig. 3B, τonset = 3.8s for both FRCRCFamide and FRCCCRFAmide). Analysis of the washout of FRCRCFamide and FRCC RFamide effects (Fig. 3C) highlighted a slower off kinetic (τoff) for FRCCRFAmide (5–6 times slower than FRCRCFAmide, Fig. 3D, τoff = 11.9s and 65.5s for FRCRCFamide and FRCCCRFAmide, respectively), pointing out the poor reversibility of this peptide's

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Cyclic CFamide, FCamide and RFamide peptides similarly potentiate ASIC3 sustained current. **A**, Typical effect of FRCRCFamide, FRCCCRFAmide, FCRRCFamide and FCRCRFamide peptides (10 µM in the pH7.4 resting solution) on rat ASIC3 activity. Currents were generated by pH drops from resting pH7.4 to pH5.5 and were recorded at –50 mV in HEK293 transfected cells. **B**, Analysis of the peak and sustained current densities elicited at pH5.5 before and after extracellular applications of peptides (sustained currents were measured 5s after initiation of ASIC3 currents, n = 9, 7, 5 and 5 for FRCRCFamide, FRCCCRFAmide, FCRRCFamide and FCRCRFamide respectively; \*p < 0.05, \*\*p < 0.01 and \*\*\*p < 0.001, Two-way ANOVA tests followed by Sidak's multiple comparisons tests). **C**, Analysis of ASIC3 current desensitization at pH5.5. Current amplitudes were measured every 100ms during the 9s following the peak, and normalized to the control peak amplitude before application of each peptide (n = 27, 9, 8, 5 and 5 for control, FRCRCFamide, FRCCCRFAmide, FCRRCFamide and FCRCRFamide, respectively; \*\*p < 0.01 and \*\*\*p < 0.0001: main effect of peptides, Two-way ANOVA followed by a Tukey's multiple comparisons test).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Comparison of the effects of FRCRCFamide and FRCCRFamide on ASIC3. A-B, Effect of FRCRCFamide or FRCCRFamide applied extracellularly for different duration (1–30s) at 10 µM in the pH7.4 resting solution. Onset kinetics of both peptides (B) were fitted over 30s using a one-phase exponential ($τ_{on}$ = 3.8s for both FRCRCFamide or FRCCRFamide, respectively; n = 9–13). C-D, Washout time analysis of the effects of FRCRCFamide and FRCCRFamide applied at 10 µM for 30s in the pH7.4 resting solution. The pH5.5 drops (10s) during the washout protocol were applied every 1 min. Off kinetics of both peptides (D) were fitted over 360s using a one-phase exponential ($τ_{off}$ = 11.9s and 65.5s for FRCRCFa and FRCCRFamide, respectively; data from 18 to 13 cells, respectively; **p < 0.01, Mixed-effects analysis to compare all points of a given condition to another). E, Dose-response curves (sigmoidal fit with Prism software) for the effects of FRCRCFamide and FRCRCFamide on pH5.5-induced ASIC3 sustained current (n = 3–21). The percentage of effect was calculated by normalizing the effect of each peptide at a given concentration by their respective effect at 30 µM.</figcaption></figure>

effect. Interestingly, the slow washout kinetic of FRCRCFamide was drastically accelerated by increasing the frequency of pH5.5 pulses during the washout protocol, whereas that of FRCRCFamide remained unchanged ($τ_{off}$ = 22.7s and 13.1 for FRCRCFamide and FRCRCFamide, respectively, see Supplementary Fig. 3), indicating more complex interactions between ASIC3 and the FRCCRFamide peptide. Finally, we performed dose-response studies for both FRCRCFamide and FRCCRFamide peptides (Fig. 3E), and found EC₅₀ values of 2.87 ± 0.44 µM and 1.19 ± 0.25 µM, respectively, indicating a similar range of apparent affinities for ASIC3.

To further investigate FRCRCFamide and FRCCRFamide effects, the question was raised to know whether cyclization, i.e. the presence of a disulfide bridge, was important for potentiating ASIC3 activity. Overall, FRCRCFamide and FRCCRFamide, i.e. without disulfide bonds, appeared to have effects very similar to those of their cyclic counterparts, with same potentiation of pH5.5-evoked sustained current (Supplementary Fig. 4A) and almost the same affinity (Supplementary Fig. 4B). The only difference between cyclic and linearized peptides was found on the washout kinetics of FRCRCFamide and FRCRCFamide, the fast washout kinetic of the cyclic peptide becoming slow for the linear peptide (Supplementary Fig. 4C). It should be noted that a small but significant proportion of the linear peptides spontaneously reformed disulfide bridges (Supplementary Fig. 4E), indicating that effects observed for FRCRCFamide and FRCCRFamide might also be due, at

least in part, to their cyclic counterparts. In any case, these data indicate that linearizing FRCRCFamide and FRCCRFamide does not seem to drastically change their potentiating effect on ASIC3 and that the slow washout kinetic appears to be characteristic of FRCRCFamide, and also potentially FRCCRFamide and FRCRCFamide, but not of FRCRCFamide, nor other previously described tetrapeptides such as FMRFamide and RPRFamide ($τ_{off}$ = 12.4 and 13.9s, respectively) (Supplementary Fig. 4C, inset).

### 3.4. FRCRCFamide and FRCCRFamide specifically potentiate the native pH5.5-induced ASIC3 current in DRG sensory neurons

The effects of FRCRCFamide and FRCCRFamide peptides were next tested on native acid-evoked currents recorded from primary culture of mouse DRG neurons (Fig. 4). Both wild-type (WT) and ASIC3⁻/⁻ (A3KO) mice were used to perform DRG neuron primary cultures, and peptides were applied onto neurons at 10 µM in the pH7.4 resting solution, for 30 s before extracellular acidification drops to pH5.5 (Fig. 4A and D). Overall analysis of the FRCRCFamide and FRCCRFamide effects on pH5.5-evoked native currents ($I_{peptide}/I_{ctrl}$) clearly showed two populations of neurons in WT DRG (Fig. 4B, dotted areas), but not in A3KO DRG (Fig. 4E). Indeed, 7 out of 15 and 5 out of 11 neurons from WT DRG had their pH5.5-evoked sustained current strongly potentiated by FRCRCFamide and FRCCRFamide, respectively (Fig. 4B–C), with also
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Effects of FRCRCFamide and FRCCRFamide on native pH5.5-evoked currents recorded in mouse DRG neurons. **A**, Typical effects of FRCRCFamide and FRCCRFamide peptides applied individually at 10 µM in the resting pH7.4 extracellular solution on different wild-type (WT) mouse DRG neurons in primary culture. Native pH5.5-evoked currents were recorded at –60 mV, according to the protocol shown by color bars above each current trace. Dotted lines indicate the zero-current level. **B**, Fold increase factors ($I_{peptide}/I_{ctrl}$) induced by FRCRCFamide and FRCCRFamide on the peak and sustained currents evoked at pH5.5 in WT DRG neurons. Two populations of neurons can be distinguished, a population in which peptides have potentiating effects (ratio ≥1.5 for the sustained current; dotted areas), and another population where they have no effect (data from 15 DRG neurons). **C**, Peak and sustained current amplitudes (pA/pF) induced in WT neurons at pH5.5, before and after applications of FRCRCFamide or FRCCRFamide. Neurons are classified depending on whether the peptides had an effect (upper panel) or not (lower panel), as indicated by the dotted areas in **C** (see black arrows). **D**, Typical effects of FRCRCFamide and FRCCRFamide applied similarly as in **A** on ASIC3⁻/⁻ (A3KO) mouse DRG neurons. **E**, Fold increase factors ($I_{peptide}/I_{ctrl}$) induced by FRCRCFamide and FRCCRFamide on the native pH5.5-evoked currents recorded from cultured A3KO neurons (data from 6 neurons). Contrary to WT neurons (**B**), only one population of neurons was found with no effect of peptides. **F**, Peak and sustained current amplitudes (pA/pF) induced in A3KO neurons at pH5.5, before and after applications of FRCRCFamide of FRCCRFamide. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

some smaller effects on the pH5.5-evoked peak current (Fig. 4B–C). The potentiated currents clearly displayed ASIC-like kinetics, with both transient peaks followed by sustained activities of varying amplitudes (Fig. 4A, upper panel). Importantly, the potentiating effects of FRCRCFamide and FRCCRFamide on these ASIC-like currents were lost in ASIC3⁻/⁻ DRG neurons (Fig. 4D–F), demonstrating the involvement of ASIC3. These results further confirm the selectivity of FRCRCFamide and FRCCRFamide toward ASIC3 among the different ASIC channel subtypes, as shown before on recombinant channels expressed in HEK293 cells (Fig. 1E), with potentiating effects observed on the pH5.5-evoked ASIC3 sustained activity. Moreover, the fact that both peptides are active on native ASIC3-type channels also suggests effects on heteromeric ASIC3 channels, which are likely to be functionally expressed in rodent DRG neurons (Deval et al., 2008; Verkest et al., 2021). On the other hand, FRCRCFamide and FRCCRFamide had no effect on WT DRG neurons showing TRPV1-like pH5.5-evoked sustained currents (Fig. 4A, lower panel).

### 3.5. In silico structural analysis and site-directed mutagenesis predict interactions of FRCRCFamide and FRCCRFamide with both the proton and non-proton binding sites of ASIC3

As FRCRCFamide and FRCCRFamide are both strong potentiators of the ASIC3 sustained activity, the effect of their combined application has then been tested (Fig. 5A). When co-applied at concentrations of 5 µM,

FRCRCFamide and FRCCRFamide generated a pH5.5-evoked sustained current modulation similar to that observed when the peptides were applied alone at 10 µM (Fig. 5A), suggesting interactions with a similar binding site. In line with these data, analysis of the washout kinetics revealed that the combination of FRCRCFamide and FRCCRFamide led to an intermediate, although not significantly different, kinetic between those of each peptide applied alone (10 µM) (Fig. 5B), in good agreement with the hypothesis of a common binding site for both peptides. In silico structural modeling was next performed to further study and predict putative interaction sites between the rat ASIC3 channel and the FRCRCFamide and FRCCRFamide peptides (Fig. 5C). The docking predicted possible binding of the peptides within both the proton and non-proton binding sites of ASIC3. Both sites were further investigated using molecular dynamics (MD) simulations. Analysis of the trajectories revealed potential interactions between the peptides and a set of amino acids, i.e. E231, E212, D252, and D358 for the proton binding site, and E247, R376, E423 and E418 for the non-proton binding site (Fig. 5C). Table 1 summarizes the strength of the interactions and suggests a stronger binding of the peptides inside the non-proton binding site (strongest interaction with E418). Interestingly, the residue E247 is the only residue showing a strong difference in interaction between the two peptides, with an absolute difference of the mean interaction energy of ~30 kcal mol⁻¹ (Table 1, Fig. 5C). This suggests a slightly different interaction on ASIC3 for the two peptides and could explain their slightly different EC₅₀ values and/or their different washout kinetics. No
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Structural elements potentially involved in the interaction of FRCRCFamide and FRCCRFamide with ASIC3. A, Combination of FRCCRFamide and FRCCRFamide at 5 µM each was tested against the application of each of them alone at a concentration of 10 µM, generating similar effects on pH5.5-evoked sustained current, without affecting the peak amplitude (n = 9–14 cells) (inset). B, Washout kinetics of the effect induced by combined application of FRCRCFamide and FRCCRFamide at 5 µM each (data from 10 cells) is compared to the application of FRCRCFamide or FRCCRFamide alone at 10 µM (same values as in Fig. 3D, \*\*p < 0.01, Mixed-effects analysis performed on all points of a given condition followed by Tukey's multiple comparisons test for a main effects of peptides). C, ProLIF ligand networks for FRCRCFamide (upper panel) and FRCCRFamide (lower panel) interactions at the proton site (left panel) and at the non-proton site (right panel) with the violin plots of the GROMACS interaction energies for the main residues (middle panel). The thickness of the dotted lines in the ligand networks are correlated to the intensity of interaction. The dotted line in the violin plot separates the residues of the proton site to the residues of the non-proton site. The peptide backbones are shown in black.</figcaption></figure>
<caption>**Table 1**</caption>

Summary of the mean interaction energy between a set of rASIC3 amino acids and both FRCRCFamide and FRCCCRFAmide peptides following the MD simulations on both the AlphaFold and the ‘post-MD’ structures. The average was taken by averaging all the interaction energies from each timestep of all 3 replicates for all 3 sites for both the proton binding sites and the non-proton binding sites using GROMACS energy ‘gmx energy’ command.

<table><thead><tr><th rowspan="3">rASIC3 residues</th><th colspan="2">FRCRCFamide</th><th colspan="2">FRCCCRFAmide</th></tr><tr><th colspan="2">Mean interaction energy<br>(kcal.mol-1 ± std)</th><th colspan="2">Mean interaction energy<br>(kcal.mol-1 ± std)</th></tr><tr><th>AlphaFold</th><th>Post-MD</th><th>AlphaFold</th><th>Post-MD</th></tr></thead><tbody><tr><td colspan="5" style="text-align: center; font-weight: bold;">Proton binding site</td></tr><tr><td>E212</td><td>-36.24 ± 17.51</td><td>-10.16 ± 9.81</td><td>-23.65 ± 14.42</td><td>-25.85 ± 14.29</td></tr><tr><td>E231</td><td>-13.25 ± 9.51</td><td>-34.68 ± 22.94</td><td>-34.13 ± 12.64</td><td>-28.01 ± 16.61</td></tr><tr><td>D252</td><td>-14.19 ± 13.98</td><td>-7.68 ± 10.79</td><td>-13.79 ± 22.02</td><td>-0.93 ± 0.89</td></tr><tr><td>D358</td><td>-5.79 ± 8.68</td><td>10.61 ± 15.07</td><td>-16.09 ± 19.19</td><td>-11.67 ± 14.91</td></tr><tr><td colspan="5" style="text-align: center; font-weight: bold;">Non-proton binding site</td></tr><tr><td>E247</td><td>-35.73 ± 7.53</td><td>-14.08 ± 16.30</td><td>-5.47 ± 12.23</td><td>-0.29 ± 0.61</td></tr><tr><td>R376</td><td>-9.93 ± 6.17</td><td>-12.77 ± 10.61</td><td>-2.93 ± 6.75</td><td>-6.48 ± 5.35</td></tr><tr><td>E418</td><td>-52.76 ± 12.66</td><td>-40.39 ± 17.23</td><td>-61.91 ± 17.72</td><td>-44.75 ± 14.73</td></tr><tr><td>E423</td><td>-27.75 ± 17.55</td><td>-21.16 ± 21.54</td><td>-22.79 ± 13.58</td><td>-26.43 ± 21.76</td></tr></tbody></table>

major differences were observed between the interactions obtained from the simulations using the AlphaFold structure or the ‘post-MD’ structure (Supplementary Fig. 5).

Point mutations in ASIC3 of the eight amino-acids potentially involved in the predicted interactions between the peptides and the channel were next designed. Negatively charged residues within the acidic pocket were mutated to positively charged ones (Arginine) (Reiners et al., 2018), except for the E212Q mutant, which was inspired by the E213Q mutation in the HyNa-5 subunit of the Hydra RFamide-peptide gated channel, a residue shown to be critical for RFamide peptide binding in this species (Ortega-Ramirez et al., 2024). In addition, residue E231 was mutated to alanine to test whether changes in the pH-dependent gating of the acidic pocket could indirectly influence peptide washout kinetics. For the non-protonbinding site, the charges of selected residues were inverted (R376D and E418R), with the exception of the E423A mutation, which was designed to perturb peptide binding without excessively impairing function, as suggested by earlier observations on the E423R mutant (Reiners et al., 2018).

All mutants were functional, except for point mutation of E247. Calculation of the sustained/peak current ratio at pH5.5 (Isst/Ipeak, Supplementary Fig. 6) suggested similar behaviors of the functional mutants comparatively to WT ASIC3, i.e. at least in response to this pH, except for the E418R mutant, which displayed a slightly higher ratio. The effects of FRCRCFamide and FRCCCRFAmide peptides were tested on the different mutants (Fig. 6), focusing on the ability of both peptides to potentiate ASIC3 activity (Fig. 6A-B and see also Table 2), but also on the washout kinetic of the FRCCCRFAmide effect (Fig. 6C). The two peptides were still able to strongly potentiate the pH5.5-evoked sustained current in the five ASIC3 mutants bearing a point mutation within the proton binding site (Fig. 6A), with fold-increase factors similar to those observed for the WT channel (Table 2). On the other hand, the sustained current potentiating effect of the two peptides was largely abolished or blunted for the E418R and E423A mutations (Fig. 6B and Table 2), within the non-proton binding site (Fig. 5B). Finally, both peptides had no effect on the amplitude of the transient peak currents generated at pH5.5 by all the mutants tested (Supplementary Fig. 7).

We next studied whether the slow washout effect of FRCCCRFAmide on ASIC3 (Fig. 3C) was affected in the proton binding site mutants (Fig. 6C). The slow τ<sub>off</sub> effect of this peptide was affected or even

abolished (i.e., becoming as fast as the one of FRCRCFamide) for the proton binding site mutants E231R and D252R, as well as E231A to a lesser extent (Fig. 6C). This may suggest that the slow washout rate of FRCCCRFAmide effect depends on additional peptide binding in the acidic pocket, or alternatively that these mutants act indirectly by altering channel gating in different ways, which can affect the peptide washout rate from the non-proton binding site by slightly modifying conformational changes. Anyway, these data indicate a more complex mechanism of action of FRCCCRFAmide with effects depending directly or indirectly on the proton binding site of ASIC3, in addition to its direct binding to the non-proton binding site.

## 4. Discussion

ASICs belong to a family of ion channels that includes the only peptide-gated channels known so far (Golubovic et al., 2007; Lingueglia et al., 1995; Schmidt et al., 2018). No mammalian peptide capable of directly opening ASICs has yet been discovered, but a relatively large number of studies report that these channels have a particular sensitivity to peptides, as they are positively modulated by FMRFamide-related peptides and dynorphins (for review, see (Vick and Askwith, 2015)). In line with these studies, a recent screening of 109 neuropeptides revealed no direct agonist of ASICs, nor any new mammalian peptide modulator of these channel, suggesting that most natural short peptides acting on ASICs are probably already known (Vyvers et al., 2018). The present study describes the positive modulation of ASIC3 activity by synthetic cyclic amidated hexapeptides that were initially synthesized as NCX inhibitors 30 years ago (Khananshvili et al., 1995), including not only peptides ending by the RFamide motif, but also peptides ending by CFamide or FCamide motifs. We particularly focused on two of them, a CFamide one (FRCRCFamide) and a RFamide one (FRCCCRFAmide), which show similar specificity and effects on ASIC3, but through slightly different mechanisms. Although it has not been tested, it is highly likely, given that the effects of these two hexapeptides are generally comparable with those of other RFamide peptides acting on ASIC3, that they also depend on the C-terminal amide group.

Both FRCRCFamide or FRCCCRFAmide strongly potentiate the pH-induced ASIC3 activity by increasing/slowing-down its desensitization current, generating a sustained activity, as already reported for other
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Effects of point mutations in ASIC3 of the amino-acids potentially involved in the interactions with FRCRCFamide and FRCCRFamide on the potentiating effect and the washout kinetic. Current densities of the pH5.5-evoked sustained currents measured in HEK293 cells transfected with different ASIC3 point mutants (holding potential –50 mV). The different bargraphs represent the current densities in pA/pF as well as the fold increase factors (arrows) induced by the two peptides. A, ASIC3 point mutations within the proton binding site (n = 6–11, One-way ANOVA tests followed by Sidak's multiple comparison tests). B, ASIC3 point mutations within the non-proton binding site (n = 4–11, One-way ANOVA tests followed by Sidak's multiple comparison tests). C, Washout kinetics of the effect of FRCCRFamide peptide on ASIC3 mutants. The washout time of the FRCCRFamide effects on six different ASIC3 mutants are represented as a function of time (WT ASIC3 channel is also represented in purple dashed line for comparison, same data as in Figs. 3D and 5B). The washout kinetics were fitted over 120s using a one-phase exponential (τoff = 24.1s for E231R, 20.6s for D252R, 37.3s for E231A, 74.1s for E212Q, 73.9s for D358R and 69.2s for WT ASIC3, n = 4–18, *p* < 0.05, Mixed-effects analysis performed on all points of a given condition followed by Dunnett's multiple comparisons test for a main effect of mutants). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

RFamide peptides. On the other hand, we did not find that these peptides were capable of significantly potentiating the ASIC3 peak current. The very fast activation kinetics of ASIC3 could prevent an underestimation of the peak current resulting from desensitization occurring before activation, as for ASIC1a (Grunder and Pusch, 2015), resulting in the absence of peak current increase in the presence of peptides. The two peptides show similar effect amplitude and EC₅₀ (~3 µM and ~1 µM, respectively), with a good selectivity toward ASIC3, at least among

ASICs, since FRCRCFamide had no effect on ASIC1a, ASIC1b, ASIC2a or ASIC2b homomeric channels. Such a selectivity for ASIC3 was further confirmed by experiments performed in cultured mouse DRG neurons, where the effects of FRCRCFamide and FRCCRFamide on native pH5.5-induced ASIC currents were lost in neurons from ASIC3⁻/⁻ mice, although more extensive experiments would be needed to study these effects across a larger DRG neuron diversity. These experiments in DRG neurons also suggest that the two peptides may not only target ASIC3
<caption>**Table 2**</caption>

Effect of FRCRCFamide and FRC&#x0305;CRFamide on the pH5.5-evoked sustained currents (measured 5s after current onset, see Fig. 1A) generated by wild-type (WT) and ASIC3 point mutants. The point mutations affecting the ASIC3 proton binding site are highlighted in orange, whereas those affecting the non-proton binding site are in green. The potentiating effects of the peptides is calculated as the fold increase of the sustained current (*p* < 0.05 and ***p* < 0.001, unpaired t-test).

<table><thead><tr><th colspan="3">FR&#x0305;CRCFamide</th><th colspan="3">FR&#x0305;CRFamide</th></tr><tr><th>ASIC3 type</th><th>Mean fold increase (I/Ictrl)</th><th>Significance (WT vs. mutant)</th><th>n</th><th>Mean fold increase (I/Ictrl)</th><th>Significance (WT vs. mutant)</th><th>n</th></tr></thead><tbody><tr><td>WT</td><td>34.08</td><td>-</td><td>6</td><td>24.22</td><td>-</td><td>8</td></tr><tr><th colspan="7">Proton binding site</th></tr><tr><td>E212Q</td><td>36.1</td><td>ns</td><td>12</td><td>36.99</td><td>ns</td><td>9</td></tr><tr><td>E231R</td><td>29.37</td><td>ns</td><td>10</td><td>21.13</td><td>ns</td><td>9</td></tr><tr><td>E231A</td><td>19.86</td><td>ns</td><td>5</td><td>36.71</td><td>ns</td><td>6</td></tr><tr><td>D252R</td><td>247.36</td><td>ns</td><td>10</td><td>89.52</td><td>ns</td><td>9</td></tr><tr><td>D358R</td><td>35.54</td><td>ns</td><td>5</td><td>44.69</td><td>ns</td><td>5</td></tr><tr><th colspan="7">Non-proton binding site</th></tr><tr><td>R376D</td><td>100.44</td><td>ns</td><td>7</td><td>10.2</td><td>ns</td><td>8</td></tr><tr><td>E418R</td><td>1.50</td><td>*** (decreased)</td><td>6</td><td>1.70</td><td>** (decreased)</td><td>5</td></tr><tr><td>E423A</td><td>1.17</td><td>*** (decreased)</td><td>9</td><td>3.83</td><td>*</td><td>11</td></tr></tbody></table>

homomeric, but also heteromeric channels that are largely expressed in DRG neurons (Verkest et al., 2021). The small effects on the pH5.5-evoked peak current seen for native channels in DRG neurons, but not for recombinant homomeric channels, could also be related to the heteromeric nature of most ASIC3 channels in sensory neurons, although we cannot exclude that it could be masked in HEK293 cells due to a higher expression level. Moreover, the two peptides do not modulate non-ASIC, TRPV1-like pH5.5-induced sustained currents (Tominaga et al., 1998) recorded in DRG neurons.

FRCRCFamide seems to act on the close state of ASIC3 channel, which is most probably also the case of FRC&#x0305;CRFamide, like all the other RFamide-related peptides. Indeed, all these peptides exert their effect on ASIC3 when applied extracellularly in the resting pH7.4 solution (Askwith et al., 2000; Catarsi et al., 2001; Deval et al., 2003; Reimers et al., 2017). In line with this interaction mode, FRCRCFamide has no effect when applied in the acid test solution, both at pH5.5 and less acidic pH values, and it has no supplemental effect when co-applied both in the resting pH7.4 plus the acid pH5.5 solutions. However, we cannot exclude that they can also bind to the open state in order to stabilize the open conformation and delay channel desensitization, but that binding is too slow to have any effect when co-applied with acidic pH, as proposed by Reimers and colleagues (Reimers et al., 2018). Moreover, there is no evidence to suggest that the peptides do not bind to desensitized channels. The potentiation of ASIC3 sustained activity was, however, strongest on currents generated by relatively more acidic pH (≤pH6.6), i.e., outside the ASIC3 window current (Deval et al., 2008; Marra et al., 2016; Yagi et al., 2006), indicating a specific effect on the sustained current generated at extreme pH by particular ASIC3 structural elements (Salinas et al., 2009), instead of an increase of the window current (Marra et al., 2016). If FRCRCFamide and FRC&#x0305;CRFamide share numerous similarities on the way they potentiate ASIC3 activity (almost the same EC₅₀, amplitude effect and τon kinetic), we found that FRC&#x0305;CRFamide has a much more slower washout kinetic (τoff ~65s, five times greater than FRC&#x0305;CRCFamide), indicating a poor reversibility for the effect of this peptide. RFamide peptides have already been proposed to act on ASIC3 by interacting with the non-proton binding site of the channel using covalent binding of a UV-sensitive RPRFamide variant, in silico docking and mutagenesis (Reimers et al., 2018). Binding of

RFamide peptides to the central vestibule of ASIC1a has also been proposed by combined docking and functional experiments on mutant channels (Bargeton et al., 2019), and cysteine-substitution mutagenesis (Frey et al., 2013). Our data on FRCRCFamide and FRC&#x0305;CRFamide are in good agreement with such an interaction, i.e., data combining FRCRCFamide and FRC&#x0305;CRFamide suggest an interaction with the same site, and effects of both peptides are lost or significantly blunted by mutations in the non-proton binding site. However, the poor reversibility of FRC&#x0305;CRFamide is eliminated by mutations within the proton binding site of ASIC3, which suggests a more complex mechanism of action for this peptide compared with FRCRCFamide. It is possible that while both peptides can interact with the non-proton binding site, the FRC&#x0305;CRFamide peptide could additionally interact directly or indirectly with the proton binding site, which influences its kinetic of dissociation from the non-proton binding site. Docking and molecular dynamics simulations suggest a possible direct interaction of FRC&#x0305;CRFamide, but also FRCRCFamide, with the proton binding site, although weaker than for the non-proton binding site. However, only FRC&#x0305;CRFamide has a slow washout kinetic. In addition, although those two peptides are supposed to bind into the acidic pocket (computational modelling), none of them affects the transient peak current, which would be expected from the molecular changes associated with proton activation of ASICs such as the collapse of the acidic pocket (Yoder et al., 2018). Although binding of neuropeptides into the acidic pocket of ASICs has already been described for dynorphin and ASIC1a (Leisle et al., 2021), it is more likely that all the RFamide and CFamide peptides described here only bind to the non-proton binding site of ASIC3. Conformational constraints for some of them, as suggested for instance by modelling data showing a strong difference in how FRC&#x0305;CRFamide and FRC&#x0305;CRFamide interact with residue E247, result in slow washout kinetics. This slow kinetics can, however, be overcome by indirect gating-related effects associated with the proton binding site when this site is mutated.

In summary, the present study identifies a set of short cyclic amided hexapeptides as new ASIC3 ligands, including peptides ending by RFamide but also CFamide and FCamide, with strong potentiating effects of the channel activity. Focusing our investigations on FRC&#x0305;CRCFamide and FRC&#x0305;CRFamide led us to identify similarities in these peptide
effects, in line with effects already reported for other peptides of the
RFamide family, including a possible interaction with the non-proton
binding site. An important point of our study could be the identifica-
tion of some short amidated peptides with long lasting effects (poor
reversibility of FRCCRFamide) providing new potential pharmacolog-
ical tools, and suggesting a more complex mechanism of action than the
one previously describes for other RFamide peptides, which could also
involve a regulation through the acidic pocket of ASIC3. This raises the
possibility of a direct binding of RFamide peptides to the acidic pocket of
ASIC channels, or more probably of a cross-talk of the binding site and
the acidic pocket. Interestingly, recent data have shown that the ligand-
binding pocket in the HyNaC channels from the cnidarian *Hydra mag-
nipapillata* that are activated by Hydra RFamide peptides is localized in
the same region as the acidic pocket of ASICs (Ortega-Ramirez et al.,
2024).

CRediT authorship contribution statement

**Maurizio Toft:** Methodology, Investigation, Formal analysis. **Maëva Meynier:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Hélène Lubrano Di Scampamorte:** Methodology, Investigation, Formal analysis. **Cédric Vallée:** Methodology, Investigation, Formal analysis. **Miguel Salinas:** Methodology, Investigation, Conceptualization. **Peijun Zhang:** Conceptualization. **Jessica Tacco:** Investigation, Formal analysis. **Anne-Sophie Gay:** Investigation, Formal analysis. **Emmanuel Bourinet:** Supervision, Conceptualization. **Eric Lingueglia:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Emmanuel Deval:** Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Data availability statement

The experimental data are available in open access in Zenodo
https://doi.org/10.5281/zenodo.17986710.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Emmanuel Deval reports financial support was provided by French
National Research Agency. Emmanuel Deval reports financial support
was provided by Foundation for Medical Research. Emmanuel Deval
reports financial support was provided by AFM-Téléthon. If there are
other authors, they declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgements

We thank Drs A. Baron, S. Diochot and J. Noel for helpful discussions, V. Friend and V. Thieffin for technical support, and J. Thoral for secretarial assistance. This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (Inserm), the Association Française contre les Myopathies (AFM grant #23731), the Agence Nationale de la Recherche (ANR-17-CE16-0018 and ANR-22-CE16-0006), the Institut ANAL-GESIA/SFETD (Société Française d'Etude et de Traitement de la Douleur), the LabEx ICST (ANR-11-LABX-0015-01), the Fondation pour la Recherche Médicale (FRM grant #FDT202404018117), the University Côte d'Azur (UniCA) and the French government, through the UCAJEDI Investments in the Future project managed by the ANR with the reference number ANR-15-IDEX-01.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.neuropharm.2025.110813.

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