Abstract
Fast inactivation is a key feature of voltage-gated sodium channels and is pivotal for countless physiological functions. Despite the prevalence of the canonical ball-and-chain model, more recent structural results suggest that fast inactivation requires multiple conformational changes beyond the binding of the inactivation particle, the IFM motif. Combining ionic current, gating current, and fluorescent measurements, here we showed that a double mutant at the bottom of the pore domain (CW) removes fast inactivation by interrupting the communication of the IFM motif and the pore. Instead of triggering fast inactivation, the IFM motif binding in CW allows the channel to enter an alternative open state. This alternative open state severely influenced the voltage sensor movements and was not accessible to wild type or other fast inactivation-deficient channels. Our results highlight the multistep nature of the fast inactivation process in mammalian voltage-gated sodium channels and demonstrate that CW modifies the channel behaviors more profoundly than simple removal of fast inactivation.