Spider toxin HM-3 differently interacts with voltage-sensing domains of Nav1.4 and blocks gating pore currents underlying periodic paralysis
Shenkarev ZO1, Lyukmanova EN1,2
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia
2Biological Faculty, Lomonosov Moscow State University, Moscow, 119234 Russia
Voltage-gated Na+ (NaV) channels contain domains that have discrete functionalities. The central pore domain allows current flow and provides ion selectivity, whereas peripherally located four voltage-sensing domains (VSD-I/IV) are needed for voltagedependent gating. Certain mutations trigger a leak current through VSDs leading to various diseases. For example, hypokalemic periodic paralysis (HypoPP) type 2 is caused by mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel NaV1.4. The gating modifier toxin Hm-3 (crab spider Heriaeus melloteei) inhibits leak (gating pore) currents through such mutant channels and represents useful hit for HypoPP therapy.
To investigate molecular basis of Hm-3 interaction with NaV1.4 channel, we studied isolated VSD-I and VSD-II by NMR in membrane mimicking environment. Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and interacts with both VSDs by the prolonged positively charged beta-hairpin. The toxin binds to different sites on the domains. On VSD-I Hm-3 interacts with the S3b helix and S3–S4 extracellular loop forming two salt bridges with conserved E208 and D211 residues, while on VSD-II the toxin binds to the S1-S2 extracellular loop interacting with E604 and D606 side chains. Nevertheless, in the both cases the allosteric changes in S4 helix conformation induced by the bound toxin block the gating pore currents. In the obtained complexes, the toxin forms lot of the stabilizing contacts with the lipids surrounding the VSDs. This suggests membrane-mediated mechanism of Hm-3/NaV1.4 interaction.
The work was supported by the Russian Science Foundation grant #24-00-000000.