Differential Na⁺ chelation underlies opposite effects of aconitine and bulleyaconitine on sodium channel permeation

Voltage-gated sodium (Nav) channel site-2 diterpenoid alkaloids, including potent toxin aconitine (ACO), are generally considered gating modifiers that stabilize activated/open conformations and impair fast inactivation. In contrast, the structurally related compound bulleyaconitine (BACO), which is used clinically in China, produces antagonist-like suppression of Na⁺ current. Compared with BACO, ACO has two additional hydroxyl groups and lacks a 4-methoxy substituent at aromatic ring. Atomic mechanisms underlying the opposite effects of BACO and ACO are unknown. In cryoEM structure 7w77, a BACO-related alkaloid (BACORA) binds in fenestration I/II of Nav1.3, exposing tertiary nitrogen toward the pore lumen. Here, BACO and ACO were docked into the channel. Monte Carlo energy minimizations (MCMs) yielded alkaloids’ binding poses located akin, but not identical to that of BACORA. MCM profiles of a hydrated Na⁺ pulled through the pore in the presence of protonated alkaloids reveal electrostatic barriers that would block ion permeation. In contrast, in the presence of unprotonated ACO, hydrated Na⁺ was transiently chelated by nitrogen and two oxygen atoms and would pass by the toxin. In BACO, the absence of hydroxyl groups near nitrogen decreases the probability of Na⁺ chelation and would shift the equilibrium of H⁺/Na⁺ exchange at nitrogen toward the protonated state that would block ion conduction. These results suggest that stabilization of the open activation gate can be mechanistically decoupled from ion permeation when a ligand exposes protonated nitrogen to the pore lumen, creating a conduction barrier. The proposed mechanism of Na⁺ permeation through ACO-bound channel is analogous to that described for the Nav1.5 channel with batrachotoxin (BTX) in fenestration III/IV. Together, these findings explain the opposite effects of ACO and BACO on Nav channels and highlight analogous mechanisms of Nav activation by ACO and BTX despite their binding in opposite fenestrations.