Kunitz-type serine protease inhibitor APEKTx1 is a peptide toxin derived from the sea anemone Anthopleura elegantissima. This toxin has a dual function, acting both as a serine protease inhibitor and as a selective and potent pore blocker of K<sub>v</sub>1.1, a shaker related voltage-gated potassium channel.
APEKTx1 is a potent toxin purified from the sea anemone A. elegantissima. Besides APEKTx1, other toxins such as APETx1, APE1-1, APE1-2, APE2-2, ApC, and APETx2 have been identified in A. elegantissima.
This peptide has 65 amino acids crosslinked by 3 disulphide bridges, and has a molecular mass of 7475 Da. It acts as a monomer. The toxin belongs to the type 2 sea anemone peptides targeting voltage-gated K channels. Other type 2 toxins are the kalicludines from Anemonia sulcata, which selectively block K<sub>v</sub>1.2 channels, and SHTX II from Stichodactyla haddoni. Structural homology is also shared with the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins (DTX I and ñ-DTX), which are potent inhibitors of voltage-gated potassium channels.
APEKTx1 is a highly selective blocker of the voltage-gated potassium channel K<sub>v</sub>1.1 with no effect on other tested potassium channels (K<sub>v</sub>1.2, K<sub>v</sub>1.3, K<sub>v</sub>1.4, K<sub>v</sub>1.5, K<sub>v</sub>1.6, Shaker IR, K<sub>v</sub>2.1, K<sub>v</sub>3.1, K<sub>v</sub>4.2 and K<sub>v</sub>4.3). APEKTx1 selectively blocks K<sub>v</sub>1.1 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<sub>v</sub> channels (kalicludines and SHTX II). APEKTx1 is thought to interact with K<sub>v</sub>1.1 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<sub>v</sub>1.1. These residues are located in the H5-loop between the S5 and S6 domains and are part of the channelâÂÂs pore.
In addition, APEKTx1 acts as a potent trypsin inhibitor (K<sub>d</sub>= 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.
APEKTx1 works by blocking the K<sub>v</sub>1.1 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<sup>+</sup> 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<sub>v</sub>1.1 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 ñ 1.0 àthat interacts with the aromatic residues on the P-loop participating in the channel-toxin interaction affinity. APEKTx1âÂÂs dyad is formed by Arg15 and Phe13, separated by 6.2 à, contributing to the formation of a physical barrier that opposes the K<sup>+</sup> efflux. Presumably, the side chain of Arg15 enters the K<sub>v</sub>1.1 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<sub>v</sub>1.1 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).