Acid-sensing ion channels (ASICs) are a novel family of non-voltage gated, pH-sensitive sodium ion channels. They are distributed in the central and peripheral nervous system of chordates and play a role in pain perception. The ASIC3 subtype in particular appears to be a key mediator of inflammatory pain. The only known selective blocker of ASIC3 is the sea anemone peptide APETx2, which blocks homo- and heteromeric ASIC3 channels. APETx2 abolishes acid-induced pain in rats and hence it provides a lead for the development of novel analgesics that target ASIC3. However, very little is known about the mechanism of action of APETx2, including the molecular basis of its interaction with ASIC3.
To address these questions, I developed a method for producing APETx2 using solid-phase peptide synthesis and native chemical ligation (NCL). NCL provides an efficient route for synthesis of long disulfide-rich peptides by splitting the peptide into smaller fragments that can subsequently be ligated. Two APETx2 fragments (APETx2_1-19 and APETx2_20-42) were synthesised and ligated, and conditions were determined to produce the correctly folded peptide. The folded synthetic toxin was shown to be equipotent with native toxin in blocking homomeric ASIC3 channels. The efficient synthesis of APETx2 laid the foundation to carry out structure-activity studies, which have greatly added to our knowledge of the toxin’s interaction with ASIC3.
A solution structure of APETx2, with substantially higher resolution and precision than previously published, was determined and used for comparing the structural integrity of all mutants made and as a template for accurately mapping the pharmacophore of the peptide.
APETx2-alanine mutants were investigated by NMR to confirm that they were correctly folded, and subsequent electrophysiology-based activity assays revealed the role of the mutated residues. Thr2, Phe15, Tyr16, Phe33 and Leu34 were shown to be the most important residues for interaction with ASIC3, with a lesser contribution from Arg17 and Tyr32 and a minor role for Pro18 and Arg31.
The key functional residues identified from mutational studies were used in combination with the newly determined high-resolution structure of APETx2 in restraint-based docking of APETx2 to a homology model of rat ASIC3. Docking was restricted to clefts and pockets (of sufficient size to accommodate the interaction surface of the toxin) along the subunit interface of the channel and the resultant model suggests that APETx2 binds to the acidic pocket region.
The stability of APETx2 was also explored via synthesis of cyclic versions of the peptide. Three cyclic analogous were made with a 6-, 7- and 8-residue linker connecting the N- and Ctermini. Cyclisation dramatically improved the biological stability of APETx2 and NMR data iii indicated that cyclisation did not perturb its structure. However, the cyclic analogs were ~100-fold less potent than wild-type APETx2 in inhibiting ASIC3. Subsequent analysis of N- and C-terminal truncation mutants revealed that both N- and C-terminal residues, particularly those at the N-terminus, are important for the APETx2 inhibition of ASIC3, thereby explaining the drop in activity for the cyclic APETx2 analogues.
The residues on ASIC3 that interact with APETx2, which were identified in the docking simulation, will guide future mutational studies of the ion channel in order to refine our understanding of the APETx2:ASIC3 interaction. This knowledge will facilitate the rational design of ASIC3 ligands with improved potency, selectivity, and therapeutic potential.