APEKTx1

Target

APEKTx1 is a highly selective blocker of the voltage-gated potassium channel K_v1.1 with no effect on other tested potassium channels (K_v1.2, K_v1.3, K_v1.4, K_v1.5, K_v1.6, Shaker IR, K_v2.1, K_v3.1, K_v4.2 and K_v4.3). APEKTx1 selectively blocks K_v1.1 channels with an IC50 value of 0.9 nM, which makes it between a 700 to 3000 times more potent inhibitor than the two known sea anemone peptides targeted against K_v channels (kalicludines and SHTX II). APEKTx1 is thought to interact with K_v1.1 through the aliphatic residue alanine (A352), an acidic residue glutamate (E353), and an aromatic residue tyrosine (Y379), as a mutation in these sites causes a loss in affinity of the toxin for K_v1.1. These residues are located in the H5-loop between the S5 and S6 domains and are part of the channel’s pore.

In addition, APEKTx1 acts as a potent trypsin inhibitor (K_d= 124 nM), probably a competitive one. However, trypsin inhibition is more potent (as it has a higher affinity) in BPTI, which can be explained by the presence of Phe13 and Pro19 in APEKTx1, causing an unfavorable interaction.

Mode of action

APEKTx1 works by blocking the K_v1.1 channel in its open conformation, showing no effect on the voltage-dependence of channel gating. In contrast to certain homologous toxins like DTX-K, APEKTx1 can completely block the K⁺ current. The binding site of the toxin is presumed to be located at the extracellular side, as evidenced by the rapid inhibition of current through K_v1.1 channels and the reversible binding upon washout. Unlike the irreversible binding of DTX K dendrotoxins (which are homologous to APEKTx1), APEKTx1's action is reversible. This is possibly due to the substitution of a lysine (either Lys3 or Lys26) with a neutral arginine in APEKTx1. APEKTx1 likely contains a dyad: a pair of amino acid residues composed of a basic residue and a hydrophobic residue separated from each other by 6.6 ± 1.0 Å that interacts with the aromatic residues on the P-loop participating in the channel-toxin interaction affinity. APEKTx1’s dyad is formed by Arg15 and Phe13, separated by 6.2 Å, contributing to the formation of a physical barrier that opposes the K⁺ efflux. Presumably, the side chain of Arg15 enters the K_v1.1 pore and interacts with the Asp377 residues, while Phe13 interacts with hydrophobic residues, including Tyr375, which is critically involved in the interaction affinity with the sea anemones toxins.

The interaction of APEKTx1 with K_v1.1 channels follows the kinetic behavior of a bimolecular reaction, meaning that the rate of binding depends on the concentration of both reactants (toxin and channel).