No abstract

Currently, five peptide modulators of acid-sensing ion channels (ASICs) attributed to structural class 1b of sea anemone toxins have been described. The APETx2 toxin is the first and most potent ASIC3 inhibitor, so its homologs from sea anemones are known as the APETx-like peptides. We have discovered that two APETx-like peptides from the sea anemone Heteractis crispa, Hcr 1b-3 and Hcr 1b-4, demonstrate different effects on rASIC1a and rASIC3 currents. While Hcr 1b-3 inhibits both investigated ASIC subtypes with IC50 4.95 ± 0.19 μM for rASIC1a and 17 ± 5.8 μM for rASIC3, Hcr 1b-4 has been found to be the first potentiator of ASIC3, simultaneously inhibiting rASIC1a at similar concentrations: EC50 1.53 ± 0.07 μM and IC50 1.25 ± 0.04 μM. The closest homologs, APETx2, Hcr 1b-1, and Hcr 1b-2, previously demonstrated the ability to inhibit hASIC3 with IC50 63 nM, 5.5, and 15.9 μM, respectively, while Hcr 1b-2 also inhibited rASIC1a with IC50 4.8 ± 0.3 μM. Computer modeling allowed us to describe the peculiarities of Hcr 1b-2 and Hcr 1b-4 interfaces with the rASIC1a channel and the stabilization of the expanded acidic pocket resulting from peptides binding which traps the rASIC1a channel in the closed state.

Toxins modulating NaV channels are the most abundant and studied peptide components of sea anemone venom. Three type-II toxins, δ-SHTX-Hcr1f (= RpII), RTX-III, and RTX-VI, were isolated from the sea anemone Heteractis crispa. RTX-VI has been found to be an unusual analog of RTX-III. The electrophysiological effects of Heteractis toxins on nine NaV subtypes were investigated for the first time. Heteractis toxins mainly affect the inactivation of the mammalian NaV channels expressed in the central nervous system (NaV1.1–NaV1.3, NaV1.6) as well as insect and arachnid channels (BgNaV1, VdNaV1). The absence of Arg13 in the RTX-VI structure does not prevent toxin binding with the channel but it has changed its pharmacological profile and potency. According to computer modeling data, the δ-SHTX-Hcr1f binds within the extracellular region of the rNaV1.2 voltage-sensing domain IV and pore-forming domain I through a network of strong interactions, and an additional fixation of the toxin at the channel binding site is carried out through the phospholipid environment. Our data suggest that Heteractis toxins could be used as molecular tools for NaV channel studies or insecticides rather than as pharmacological agents.

Parkinson’s disease (PD) is a socially significant disease, during the development of which oxidative stress and inflammation play a significant role. Here, we studied the neuroprotective effects of four Kunitz-type peptides from Heteractis crispa and Heteractis magnifica sea anemones against PD inductors. The peptide HCIQ1c9, which was obtained for the first time, inhibited trypsin less than other peptides due to unfavorable interactions of Arg17 with Lys43 in the enzyme. Its activity was reduced by up to 70% over the temperature range of 60–100 °C, while HCIQ2c1, HCIQ4c7, and HMIQ3c1 retained their conformation and stayed active up to 90–100 °C. All studied peptides inhibited paraquat- and rotenone-induced intracellular ROS formation, in particular NO, and scavenged free radicals outside the cells. The peptides did not modulate the TRPV1 channels but they affected the P2X7R, both of which are considered therapeutic targets in Parkinson’s disease. HMIQ3c1 and HCIQ4c7 almost completely inhibited the ATP-induced uptake of YO-PRO-1 dye in Neuro-2a cells through P2X7 ion channels and significantly reduced the stable calcium response in these cells. The complex formation of the peptides with the P2X7R extracellular domain was determined via SPR analysis. Thus, these peptides may be considered promising compounds to protect neuronal cells against PD inductors, which act as ROS production inhibitors and partially act as ATP-induced P2X7R activation inhibitors.