Fig. 9: Mechanism.

In the Ca²⁺-free closed state, constrictions in the narrow neck and extracellular vestibule limit the access of either anions or the blocker 1PBC, whose binding site is occupied by Tyr 514 on α3. Ca²⁺ binding results in a series of transitions in the channel that opens the pore by rearranging the outer vestibule. The outward movement of α4 widens the outer pore entrance, while the more extended conformational change of α3 relocates Tyr 514 away from the pore and projects the adjacent Arg 515 towards the pore lumen, creating a site that accommodates the blocker. These rearrangements are subsequently propagated to the intracellular part of the narrow neck region to release a hydrophobic gate that stabilizes the constricted pore in the closed state. The binding of the blocker to the site immediately external to the narrow neck results in a direct blockade of the ion conduction path, thereby inhibiting channel activity. Blocker access to a pre-open conformation, where the site is already remodeled but the gate is still closed, appears to be feasible and might be represented in the observed structure.