Short RFamide and CFamide peptides as novel positive modulators of Acid-Sensing Ion Channel 3 with similar potentiating effects but different reversibility

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 have kept sensitivity to peptides and are notably modulated by the molluscan FMRFamide and other related mammalian neuropeptides ending by the RFamide motif. Screening efforts have been made to identify and characterize natural peptides able to modulate ASICs’ activity, and some peptides from different species are already known to modulate ASIC1a, ASIC1b and ASIC3. We identified here a set of synthetic short amidated hexapeptides, initially designed thirty years ago for their ability to inhibit the Na/Ca exchanger, as potent and selective positive modulators of the ASIC3 acid-induced activity. We focused on two of them, a RFamide peptide (FR CC RFamide) and a CFamide peptide (FRC̄R̄C̄Famide), 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 the current 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 for the FRC̄C̄RFamide peptide was much slower than those of FRC̄R̄C̄Famide 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 initially identified for the synthetic compound GMQ (2-guanidine-4-methylquinazoline), as already reported before for other RFamide peptides, but our data also suggest possible additional effects of FRC̄C̄RFamide involving directly or indirectly the proton binding domain. These findings expand our understanding of peptide modulation of ASIC channels and identify novel pharmacological tools selective among ASICs for investigating ASIC3 function.