Structural Studies of the Interaction Between the Paddle Motif from NaV1.5Div and the Sea Anemone Toxin Anthopleurin-A

Abstract

Voltage gated sodium channels (VGSC) are membrane proteins that are important in the central and peripheral nervous systems and cardiac muscles among others. The main function of VGSC is in the propagation of electrical signals by depolarizing excitable cells. Numerous diseases have been linked to defects in VGSC including epilepsy, mental retardation, and several ailments that could lead to sudden cardiac death. Furthermore, these channels are one of the primary targets of toxins from venomous animals. These toxins have been used as an excellent probe to study the function of VGSC due to their specific and potent effect on VGSC. The sea anemone toxin anthopleurin-A (ApA) inhibits the fast inactivation of the cardiac channel NaV1.5 by binding to the S3b-S4a motif (paddle motif) on the voltage sensing domain (VSD) of DIV. Here we used NMR spectroscopy to determine the solution structures of NaV1.5 DIV paddle motif and of ApA toxin, both in DPC micelles. The structure of the paddle motif takes a helix-turn-helix motif that aligns well with the cryo-EM structure of the full channel. Through paramagnetic relaxation enhancement, we determined that the paddle motif is mainly interacting with the interface region of the micelle. Based on 15N NMR relaxation studies, the paddle motif is mostly rigid with some residues at and around the loop in addition to the C-terminus undergoing ps-ns motion. NMR titration experiments between a 15N labeled paddle motif with unlabeled ApA toxin uncovered specific residues on the paddle that likely interact with the toxin. Further electrophysiology studies are needed to confirm the importance of these residues in toxin binding. We are in the process of determining the three-dimensional structure of the toxin-paddle complex in micelle.