Roscioni, A.; Alberini, G.; Miceli, F.; Benfenati, F.; Taglialatela, M.; Maragliano, L.

Gain-of-function (GoF) variants in the Kv7.2 channel are associated with a clinically relevant subset of neurodevelopmental disorders. While most GoF substitutions identified so far affect the voltage-sensing domain, we recently described three mutations in the intracellular-facing activation gate (AG), G313S, A317T, and L318V. Electrophysiological recordings showed that these variants increase macroscopic current density and enhance channel open probability. Consistently, molecular dynamics (MD) simulations revealed that they hinder complete channel closure by destabilizing the closed AG and increasing hydration of the central cavity (CC). Whether this partially open conformation can support K^+ permeation, however, remained an open question. Here, we combined long-timescale atomistic simulations and simulations with applied electric fields to evaluate the stability of these mutant-associated AG states over longer timescales and their functional relevance. In new trajectories, all three substitutions consistently shifted the closed intracellular gate toward a widened, water-accessible conformation, accompanied by increased CC hydration. We then assessed the functional significance of this partially open state by simulating the A317T channel under applied electric fields. The conformation supported K^+ translocation, whereas the closed WT pore remained impermeable under all tested voltages. When simulations were started from open channel conformations, both WT and A317T conducted K^+ ions, indicating that the main effect of the substitution is to destabilize closure of the intracellular gate rather than to alter the fully conductive open state. Together, these data show that AG GoF variants can generate an intermediate gate conformation that permits ion permeation, providing a mechanistic link between mutant-induced pore remodeling and Kv7.2 dysfunction in KCNQ2-related disease.