A single conserved basic residue in the potassium channel filter region controls KCNQ1 insensitivity toward scorpion toxins

Abstract

Although many studies concerning the sensitivity mechanism of scorpion toxin-potassium channel interactions have been reported, few have explored the biochemical insensitivity mechanisms of potassium channel receptors toward natural scorpion toxin peptides, such as the KCNQ1 channel. Here, by sequence alignment analyses of the human KCNQ1 channel and scorpion potassium channel MmKv2, which is completely insensitive to scorpion toxins, we proposed that the insensitivity mechanism of KCNQ1 toward natural scorpion toxins might involve two functional regions, the turret and filter regions. Based on this observation, a series of KCNQ1 mutants were constructed to study molecular mechanisms of the KCNQ1 channel insensitivity toward natural scorpion toxins. Electrophysiological studies of chimera channels showed that the channel filter region controls KCNQ1 insensitivity toward the classical scorpion toxin ChTX. Interestingly, further residue mutant experiments showed that a single basic residue in the filter region determined the insensitivity of KCNQ1 channels toward scorpion toxins. Our present work showed that amino acid residue diversification at common sites controls the sensitivity and insensitivity of potassium channels toward scorpion toxins. The unique insensitivity mechanism of KCNQ1 toward natural scorpion toxins will accelerate the rational design of potent peptide inhibitors toward this channel.

Keywords: Insensitivity; KCNQ1; Peptide design; Potassium channel; Sensitivity.

Fig. 1

Two classical functional regions in scorpion toxin-sensitive potassium channels are also present in KCNQ1 and MmKv2, which are completely insensitive to scorpion toxins. A, KCNQ1 and MmKv2 are scorpion venom and scorpion toxin insensitivity channels. The venom of M. martensii was used to test the sensitivity of KCNQ1 and MmKv2 potassium channels, which was diluted using extracellular fluid as volume ratio 1:1 before using. B, KCNQ1 and MmKv2 belong to the same voltage-gated potassium channel subfamily. Conserved amino acid residues are indicated in bright green, and secondary structure elements are indicated above the sequences. The conserved positively charged residues in the voltage sensor of the S4 segment are marked, and the characteristic residues of the ion selectivity filter “GYGD” are also indicated. C, Sequence alignments of toxin-interacting regions (turret and filter regions) in KCNQ1 and KCNQ2 potassium channels, scorpion-derived potassium channel MmKv2, and scorpion-toxin-sensitive potassium channels, including the filter region and turret region. The complte conserved amino acid residues are indicated in blue, such as “GYGD”. Secondary structure elements are indicated above the sequences.

Fig. 2

The filter region of the KCNQ1 potassium channel determines the insensitivity of KCNQ1 toward the scorpion toxin ChTX. A, Mutant designs for the KCNQ1 channel. KCNQ1-M1 is a double region mutant, KCNQ2-M1 is a turret region mutant, and KCNQ1-M3 is a filter region mutant. B-E, Sensitivities of the KCNQ1 channel and designed channel mutants toward the natural scorpion toxin ChTX.

Fig. 3

A single basic residue in filter region determines the insensitivity of KCNQ1 channels toward scorpion toxins. A, Sequence alignment of the scorpion toxin-sensitive channel Kv1.3, scorpion toxin-insensitive channel KCNQ1, and the KCNQ1 mutant channels targeting filter region. B-D, Sensitivities of the KCNQ1 channel and designed single residue mutants toward the natural scorpion toxin ChTX.

Fig. 4

One basic residue in the potassium channel filter region abolishes the potassium channel sensitivity toward scorpion toxins. To explore its influence in detail, we named the filter region sequence GYGDKVPQ-322 (in KCNQ1 as an example) as P4′-P3′-P2′-P1′-P1-P2-P3-P4. A, Representative residues of filter regions in the scorpion toxin-sensitive and -insensitive potassium channels. B, The P1 site, located at the entrance of K⁺ ion in potassium channels, controls channel sensitivity toward scorpion toxins.