BPS2026 – Pore mutations in voltage-gated sodium channels stabilize the channels permanently in the slow inactivated state

Voltage-gated sodium (Nav) channels mediate fast inward sodium current and are main drivers for excitation. This inward sodium conductance is attenuated through two distinct inactivation mechanisms. The fast inactivation attenuates the sodium current milliseconds after activation. The slow inactivation, on the other hand, gradually inactivates the channel over a much longer period of time and sets in with fast repetitive firing. Compared to our knowledge of fast inactivation, the molecular mechanism of slow inactivation remains largely elusive. Part of the reason is that so far, no mutant in Nav channel has been found to stabilize the channel in the slow inactivated state, unlike the classical W434F mutant in the Shaker potassium channel. Here, we identified 4 large hydrophobic residues, one from each domain, in the inner cavity of the Nav channels that once mutated to alanine, stabilize the channel in the slow inactivated state. Single alanine mutants activate at more depolarized voltages and enter the slow inactivated state both faster and more completely. When combined, the identified residues work additively so that double or triple alanine mutations of the identified residues stabilize the majority of the channels in the slow inactivated state, rendering the channels almost completely nonconductive. In the most extreme case in which all 4 residues are mutated, no discernable ionic current is observed even in the presence of 120 mM external Na + while robust gating current is recorded. Compared to the wild-type, the QV curve of the quadruple mutant is ∼15 mV shifted to more depolarized voltages. Taken together, we demonstrate the importance of the pore inner cavity residues in the slow inactivation process and provide a model mutant channel to investigate slow inactivation in Nav channels. Support R01-GM030376 and QuBBE-QLCI (NSF-OMA-2121044).