The drug 4-aminopyridine (4-AP) is a Kv channel inhibitor that improves electrical conduction in demyelinated nerves and is used for the symptomatic treatment of multiple sclerosis. Despite its extensive use, the mechanism of 4-AP inhibition remains debated with competing models of closed-state binding and stabilization versus open-state block. We propose a model with an intermediate state, where 4-AP binds to a pocket that exists exclusively on the closed state but is only accessible in a closed state before channel opening (pre-open). To distinguish between these possibilities, we combined electrophysiological measurements and targeted point mutations to elucidate the mechanism of 4-AP inhibition. We observed that 4-AP decreased the total gating charge by about 10%, even when the bundle crossing gate is constitutively blocked by a bulky hydrophobic tryptophan residue that prevents potassium permeation, indicating that intracellular accessibility via the pore is not required. Using the ILT mutant we show that this reduction in the gating charge is due to the abolition of the final activation step of the gating charge that is associated with channel opening. Decreasing the volume of a residue in the S6 helices interface that is part of the putative binding site, allows for closed state binding, indicating that 4-AP diffuses through the membrane into the pore via this site. Consistent with our kinetic model, strong depolarization reduces 4-AP inhibition by promoting unbinding. After unbinding has occurred, a large hyperpolarization prevents 4-AP rebinding because it minimizes the intermediate state residence during closing. These results support that 4-AP works by binding to the pre-open state and preventing the complete activation of the VSD and thus channel opening. This work elucidates the mechanistic details of 4-AP action on Kv channels, with implications for its therapeutic applications. Support: NIH R01-GM030376.
Pinto et al. (2026) studied this question.