Voltage-Sensing Domain of the Third Repeat of Human Skeletal Muscle NaV1.4 Channel As a New Target for Spider Gating Modifier Toxins

摘要

Voltage-gated sodium channels (NaV) have a modular architecture and containfive membrane domains. The central pore domain is responsible for ionconduction and contains a selectivity filter, while the four peripheralvoltage-sensing domains (VSD-I/IV) are responsible for activation and rapidinactivation of the channel. “Gating modifier” toxins fromarthropod venoms interact with VSDs, influencing the activation and/orinactivation of the channel, and may serve as prototypes of new drugs for thetreatment of various channelopathies and pain syndromes. The toxin-bindingsites located on VSD-I, II and IV of mammalian NaV channels have beenpreviously described. In this work, using the example of the Hm-3 toxin fromthe crab spider Heriaeus melloteei, we showed the presence ofa toxin-binding site on VSD-III of the human skeletal muscle NaV1.4 channel. Adeveloped cell-free protein synthesis system provided milligram quantities ofisolated (separated from the channel) VSD-III and its 15N-labeled analogue. Theinteractions between VSD-III and Hm-3 were studied by NMR spectroscopy in themembrane-like environment of DPC/LDAO (1 : 1) micelles. Hm-3 has a relativelyhigh affinity to VSD-III (dissociation constant of the complex Kd ~6 μM),comparable to the affinity to VSD‑I and exceeding the affinity to VSD-II.Within the complex, the positively charged Lys25 and Lys28 residues of thetoxin probably interact with the S1–S2 extracellular loop of VSD-III. TheHm-3 molecule also contacts the lipid bilayer surrounding the channel.