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Front Pharmacol
2020 Apr 07;11:1010. doi: 10.3389/fphar.2020.01010.
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Tuning Scorpion Toxin Selectivity: Switching From KV1.1 to KV1.3.
Gigolaev AM
,
Kuzmenkov AI
,
Peigneur S
,
Tabakmakher VM
,
Pinheiro-Junior EL
,
Chugunov AO
,
Efremov RG
,
Tytgat J
,
Vassilevski AA
.
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Voltage-gated potassium channels (KVs) perform vital physiological functions and are targets in different disorders ranging from ataxia and arrhythmia to autoimmune diseases. An important issue is the search for and production of selective ligands of these channels. Peptide toxins found in scorpion venom named KTx excel in both potency and selectivity with respect to some potassium channel isoforms, which may present only minute differences in their structure. Despite several decades of research the molecular determinants of KTx selectivity are still poorly understood. Here we analyze MeKTx13-3 (Kalium ID: α-KTx 3.19) from the lesser Asian scorpion Mesobuthus eupeus, a high-affinity KV1.1 blocker (IC50 ~2 nM); it also affects KV1.2 (IC50 ~100 nM), 1.3 (~10 nM) and 1.6 (~60 nM). By constructing computer models of its complex with KV1.1-1.3 channels we identify specific contacts between the toxin and the three isoforms. We then perform mutagenesis to disturb the identified contacts with KV1.1 and 1.2 and produce recombinant MeKTx13-3_AAAR, which differs by four amino acid residues from the parent toxin. As predicted by the modeling, this derivative shows decreased activity on KV1.1 (IC50 ~550 nM) and 1.2 (~200 nM). It also has diminished activity on KV1.6 (~1500 nM) but preserves KV1.3 affinity as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. In effect, we convert a selective KV1.1 ligand into a new specific KV1.3 ligand. MeKTx13-3 and its derivatives are attractive tools to study the structure-function relationship in potassium channel blockers.
Figure 1. (A–C) Modeled structure of MeKTx13-3 in complex with KV1.1–1.3. (A) Overall structure of the KV1.3–MeKTx13-3 complex after 100-ns MD simulation inside a hydrated lipid bilayer membrane. Four channel α-subunits with identical sequences are color-coded. The pore domain helices of the channel subunit in the foreground and voltage-sensing domain (VSD) of the adjacent subunit, as well as extended extracellular loops of the VSDs are omitted for clarity. Lipids are shown in a semi-transparent space-filling representation; atoms are colored: oxygen, red; phosphorus, orange; nitrogen, blue; hydrogen of amino group, white; carbon of POPC, light-yellow; carbon of POPE, yellow; and carbon of cholesterol, beige. Some lipids are omitted for clarity. MeKTx13-3 is presented in pink; residue Lys26 (plugs the channel pore) is shown as sticks. (B, C) Close-up view on the channel pore vestibule area in complexes KV1.1–MeKTx13-3 and KV1.2–MeKTx13-3, respectively. Channels are shown in a semi-transparent representation. Lys26 and residues involved in the intermolecular contacts not present in the KV1.3–MeKTx13-3 complex are shown as sticks. Hydrogen bonds and salt bridges are shown as dashed yellow lines. Lipids are omitted for clarity. (D) Amino acid sequence alignment of the extracellular pore region of KV1.1–1.3 channels. Residue numbering is above each sequence; different residues are shaded gray; sequences of S5-P loops containing channel-specific residues are underlined.
Figure 2. Production of MeKTx13-3 and its derivative. (A) Amino acid sequence alignment of MeKTx13-3 and MeKTx13-3_AAAR. Gray shading indicates the positions where replacements were introduced. Cysteine residues are in bold, and lines above the sequences indicate disulfide bonds. Take a note that recombinant analogue of MeKTx13-3 does not bear the C-terminal amidation of the natural toxin. (B, C) Reversed-phase HPLC separation of recombinant MeKTx13-3 and MeKTx13-3_AAAR after digestion by enteropeptidase. For target peptides the measured molecular masses are indicated.
Figure 3. Electrophysiological profiling of MeKTx13-3 and MeKTx13-3_AAAR activities. (A) Representative traces of currents through KV1.1–1.6 in control (black) and after application of 10 nM toxin (blue). (B) Concentration–response curves of MeKTx13-3 (left) and MeKTx13-3_AAAR (right) on KV1.1–1.3 and 1.6 obtained by electrophysiological measurements. IC50 values are listed in Table 1.
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