Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family

Abstract

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 μM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.

FIGURE 1.

Primary structures of scorpion Kunitz-type toxins. Seven Kunitz-type toxins from scorpion, including four new Kunitz-type toxins; BmKTT-1, BmKTT-2, and BmKTT-3 from scorpion B. martensii, LmKTT-1c from scorpion Lychas mucronatus, and three known Kunitz toxins, LmKKT-1a, LmKTT-1b, and Hg1. Identical and similar residues are noted. The cysteine residues are marked with Roman numerals.

FIGURE 2.

Inhibitory effects of scorpion Kunitz-type toxins on trypsin. A, inhibitory effects of rHg1 peptide on trypsin with BPTI and BSA as controls. B, inhibitory effects of seven scorpion Kunitz-type toxins at different concentrations on trypsin using the same conditions. Data represent the mean ± S.E. of at least three experiments.

FIGURE 3.

Effects of seven scorpion Kunitz-type toxins on mKv1.3 channel currents. A and B, blocking effects of rHg1 on mKv1.3 K⁺ currents. C, blocking effects of rBmKKT-3 on mKv1.3 currents. D, blocking effects of rLmKTT-1a on mKv1.3 currents. E, blocking effects of rLmKTT-1b on mKv1.3 currents. F, blocking effects of rLmKTT-1c on mKv1.3 currents. G, blocking effects of rBmKTT-1 on mKv1.3 currents. H, blocking effects of rBmKTT-2 on mKv1.3 currents. I, concentration-dependent inhibition of mKv1.3 channels by rBmKTT-1. J, concentration-dependent inhibition of mKv1.3 channels by rBmKTT-2. K, concentration-dependent inhibition of mKv1.3 channels by rHg1. Data represent the mean ± S.D. of at least three experiments.

FIGURE 4.

Effects of rHg1 on other potassium channel currents. A, blocking effects of rHg1 on mKv1.1 currents. B, blocking effects of rHg1 on hKv1.2 currents. C, blocking effects of rHg1 on hSKCa3 currents. D, blocking effects of rHg1 on mBKCa currents.

FIGURE 5.

Effects of the Hg1 mutants on mKv1.3 channel currents. A–H, representative current traces of mKv1.3 channel showing the blockage of currents by Hg1 and its mutants. A, 100 nm Hg1; B, 100 nm Hg1-H2A; C, 100 nm Hg1-H3A; D, 100 nm Hg1-N4A; E, 100 nm Hg1-R5A; F, 100 nm Hg1-L9A; G, 100 nm Hg1-L10A; H, 100 nm Hg1-K13A. I, circular dichroism spectra of Hg1, Hg1-H2A, Hg1-H3A, and Hg1-N4A peptides. J, circular dichroism spectra of Hg1, Hg1-R5A, Hg1-L9A, Hg1-L10A, and Hg1-K13A peptides. deg, degrees.

FIGURE 6.

Functional importance of residues in the C-terminal region of Hg1. A–D, representative current traces of mKv1.3 channels showing the block of currents by Hg1 mutants: A, 100 nm Hg1-K56A; B, 100 nm Hg1-R57A; C, 100 nm Hg1-F61A; and D, 100 nm Hg1-K63A. E, the circular dichroism spectra analyses of Hg1, Hg1-K56A, Hg1-R57A, Hg1-F61A, and Hg1-K63A peptides. F, concentration-dependent inhibition of Kv1.3 channel currents by Hg1, Hg1-K56A, Hg1-R57A, Hg1-F61A, and Hg1-K63A peptides. Data represent the mean ± S.D. of at least three experiments.

FIGURE 7.

Interaction of Hg1 with mKv1.3 channels explored by computational simulation. A, the key residue Lys-56 was surrounded mainly by the pore region residues from Kv1.3 channels within a contact distance of 4 Å. B–D, Phe-61, Lys-63, and Arg-57 contacted the residues of mKv1.3 channels, respectively.

FIGURE 8.

Differential binding interfaces of Kunitz-type toxins blocking potassium channels. A, sequence alignments of Kunitz-type potassium channel toxins Hg1 from scorpion, δ-DTX from snake, and HWTX-XI from spider. B, the main functional residues of Kunitz-type toxin δ-DTX (structure modeled with DTX-K as template, PDB code 1DTK) interacting with the Kv1.1 channel. C, the main functional residues of Kunitz-type toxin HWTX-XI (PDB code 2JOT) interacting with the Kv1.1 channel. D, the main functional residues of Kunitz-type toxin Hg1 (structure modeled with BPTI as template, PDB code 6PTI) interacting with Kv1.3 channels.