Molecular mechanism of HNTX-I activates the intermediate-conductance Ca2+-activated K+ (IK) channels

The IK channel, a potassium ion channel regulated by calcium ions and voltages in a bidirectional manner, has been implicated in a range of diseases. However, there are currently few compounds available that can target the IK channel with high potency and specificity. Hainantoxin-I (HNTX-I) is the first peptide activator of IK channel discovered so far, but its activity is not ideal, and the underlying mechanism interaction between HNTX-I toxin and IK channel remains unclear. Thus, our study aimed to enhance the potency of IK channel activating peptides derived from HNTX-I and elucidate the molecular mechanism underlying the interaction between HNTX-I and the IK channel. By employing virtual alanine scanning mutagenesis, we generated 11 HNTX-I mutants using site-directed mutagenesis to pinpoint specific residues crucial for the HNTX-I and IK channel interaction. Subsequently, we identified key residues on the IK channel that are involved in the interaction with HNTX-I. Additionally, molecular docking was employed to guide the molecular engineering process and clarify the binding interface between HNTX-I and the IK channel. Our results demonstrate that HNTX-I primarily acts on the IK channel via the N-terminal amino acid, and its interaction with the IK channel is mediated by electrostatic and hydrophobic interactions, specifically the amino acid residues at positions 1, 3, 5, and 7 on HNTX-I. This study provides valuable insights into the peptide toxins that may serve as potential templates for the development of activators with enhanced potency and selectivity for the IK channel.