APETx1 from Sea Anemone Anthopleura elegantissima Is a Gating Modifier Peptide Toxin of the Human Ether-a-go-go- Related Potassium Channel

Abstract

We studied the mechanism of action and the binding site of APETx1, a peptide toxin purified from sea anemone, on the human ether-a-go-go-related gene (hERG) channel. Similar to the effects of gating modifier toxins (hanatoxin and SGTx) on the voltage-gated potassium (Kv) 2.1 channel, APETx1 shifts the voltage-dependence of hERG activation in the positive direction and suppresses its current amplitudes elicited by strong depolarizing pulses that maximally activate the channels. The APETx1 binding site is distinctly different from that of a pore-blocking peptide toxin, BeKm-1. Mutations in the S3b region of hERG have dramatic impact on the responsiveness to APETx1: G514C potentiates whereas E518C abolishes the APETx1 effect. Restoring the negative charge at position 518 (methanethiosulfonate ethylsulfonate modification of 518C) partially restores APETx1 responsiveness, supporting an electrostatic interaction between E518 and APETx1. Among the three hERG isoforms, hERG1 and hERG3 are equally responsive to APETx1, whereas hERG2 is insensitive. The key feature seems to be an arginine residue uniquely present at the 514-equivalent position in hERG2, where the other two isoforms possess a glycine. Our data show that APETx1 is a gating modifier toxin of the hERG channel, and its binding site shares characteristics with those of gating modifier toxin binding sites on other Kv channels.Except for a few recent cases, membrane channels have not been amenable to direct structural determination by crystallography. Peptide toxins targeting ion channels have been used to probe the conformations of their binding sites on the target channels in a so-called "peptide toxin footprinting" approach (Goldstein et al

S3b Amino Acid Substitutions and Ancillary Subunits Alter the Affinity of Heteropoda venatoria Toxin 2 for Kv4.3

“…To quantify the effect of Kv4.3 mutations on interaction with HpTx2, we determined the K d value for WT Kv4.3 and several of the Kv4.3 S3b mutants at toxin concentrations between 1 nM and 10 M HpTx2. We used a method previously used with the gatingmodifier toxins HaTx, Scodra griseipes toxin (SGTx), and APETx1 (Lee et al, 2003;Zhang et al, 2007). Like HpTx2, these toxins all interact with the S3b region of a voltagegated K ϩ channel; Kv2.1 in the case of HaTx and SGTx, Kv11.1 (human ether-à -go-go-related gene) with APETx1.…”

“…In several cases, careful channel mutagenesis studies have been performed. These studies have shown that the toxin binding sites are often complex and are dependent on either a combination of charged and hydrophobic amino acids or charge interactions alone (Swartz and MacKinnon, 1997b;Alabi et al, 2007;Catterall et al, 2007;Smith and Blumenthal, 2007;Swartz, 2007;Zhang et al, 2007;Bosmans et al, 2008;Norton and McDonough, 2008). HpTx2 is thus far unique among the ICK toxins in relying solely on hydrophobic contacts for channel interaction.…”

BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker

“…These toxins share a structural homology with the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins (from mamba snakes) which are highly potent blockers of K V channels [10,11]. The third type is composed of toxins containing 41 or 42 amino acid residues and three disulfide bridges: BDS I and II toxins (A. viridis) inhibit K V 3.1, K V 3.2 and K V 3.4 currents by acting as channel-gating modulators and APETx1 (A. elegantissima) is a gating modifier toxin of K V 10.1 and K V 11.1 [12,13]. The type 4 family is represented by two putative homologous K V channel blocking peptides, SHTX I and II, from Stichodactyla haddoni [14].…”

Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

“…For instance, the tarantula toxins JZTX-I and III bind to both Nav (see above) and Kv channels [134,145,146,149], ProTx-I interacts with Nav, Kv, and Cav channels, and ProTx-II interacts with Nav and Cav channels [148]. In addition, there are other unrelated toxins like sea-anemone toxins that affect Kv channels with a similar mechanism [151][152][153]. The amphipathic structure of the cysteine-knot toxins with a cluster of hydrophobic residues surrounded by polar residues [133] suggests that they are likely to partition into the membrane and possibly act on the VSD from there [64,[154][155][156][157].…”