A natural point mutation changes both target selectivity and mechanism of action of sea anemone toxins

Abstract

APETx3, a novel peptide isolated from the sea anemone Anthopleura elegantissima, is a naturally occurring mutant from APETx1, only differing by a Thr to Pro substitution at position 3. APETx1 is believed to be a selective modulator of human ether-á-go-go related gene (hERG) potassium channels with a K(d) of 34 nM. In this study, APETx1, 2, and 3 have been subjected to an electrophysiological screening on a wide range of 24 ion channels expressed in Xenopus laevis oocytes: 10 cloned voltage-gated sodium channels (Na(V) 1.2-Na(V)1.8, the insect channels DmNa(V)1, BgNa(V)1-1a, and the arachnid channel VdNa(V)1) and 14 cloned voltage-gated potassium channels (K(V)1.1-K(V)1.6, K(V)2.1, K(V)3.1, K(V)4.2, K(V)4.3, K(V)7.2, K(V)7.4, hERG, and the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels and provides this toxin the ability to become a potent (EC(50) 276 nM) modulator of voltage-gated sodium channels (Na(V)s) because it slows down the inactivation of mammalian and insect Na(V) channels. Our study also shows that the homologous toxins APETx1 and APETx2 display promiscuous properties since they are also capable of recognizing Na(V) channels with IC(50) values of 31 nM and 114 nM, respectively, causing an inhibition of the sodium conductance without affecting the inactivation. Our results provide new insights in key residues that allow these sea anemone toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins thus far believed to be highly selective.

International Union of Basic and Clinical Pharmacology. XCI. Structure, Function, and Pharmacology of Acid-Sensing Ion Channels and the Epithelial Na+Channel

“…On the basis of the presence of the basic-aromatic toxin surface patch it has been suggested that APETx2 may bind as PcTx1 to the acidic pocket (Baron et al, 2013). APETx2 is not selective for ASICs and inhibits voltage-gated Na + currents in DRG neurons with an IC 50 of 2.6 mM, recombinant Na v 1.8 (IC 50 of 55 nM or 19 mM, depending on the study), and Na v 1.2 (IC 50 of 114 nM) (Blanchard et al, 2012;Peigneur et al, 2012).…”

Acid-sensing ion channel (ASIC) structure and function: Insights from spider, snake and sea anemone venoms

“…The structure of APETx2 consists of a compact hydrophobic core composed of a four-stranded β-sheet containing three disulfide bonds, resembling a defensin-like fold ( Figure 2B & 3D) (Chagot et al, 2005;Jensen et al, 2014). At higher concentrations APETx2 also inhibits the voltage-gated sodium channels Na V 1.2, Na V 1.6, and Na V 1.8 with varying degrees of potency depending on subtype and study (Blanchard et al, 2012;Peigneur et al, 2012). Additional off target effects have been shown with APETx2 also inhibiting the cardiac hERG channel in the low micromolar range (Jensen et al, 2014), demonstrating that this prototypical "selective" ASIC3…”

Binding Site and Inhibitory Mechanism of the Mambalgin-2 Pain-relieving Peptide on Acid-sensing Ion Channel 1a

“…The MitTx identified from the venom of the Texas coral snake does not inhibit but potently activates several homomeric and heteromeric ASIC channels and helped to identify a role for peripheral ASIC1a-containing channels in cutaneous pain (13) and to define the structure of the open state of ASIC1a (14). The sea anemone toxin APETx2, a peptide that blocks ASIC3-containing channels (15) and inhibits to some extent Nav1.8 voltage-dependent Na ϩ channels (16,17), has been used to demonstrate the role of peripheral ASIC3-containing channels in acidic, inflammatory, and postoperative pain (18 -20).…”