Structural mechanism of voltage-gated sodium channel slow inactivation.

Voltage-gated sodium (NaV) channels mediate a plethora of electrical activities. NaV channels govern cellular excitability in response to depolarizing stimuli. Inactivation is an intrinsic property of NaV channels that regulates cellular excitability by controlling the channel availability. The fast inactivation, mediated by the Ile-Phe-Met (IFM) motif and the N-terminal helix (N-helix), has been well-characterized. However, the molecular mechanism underlying NaV channel slow inactivation remains elusive. Here, we demonstrate that the removal of the N-helix of NaVEh (NaVEhΔN) results in a slow-inactivated channel, and present cryo-EM structure of NaVEhΔN in a potential slow-inactivated state. The structure features a closed activation gate and a dilated selectivity filter (SF), indicating that the upper SF and the inner gate could serve as a gate for slow inactivation. In comparison to the NaVEh structure, NaVEhΔN undergoes marked conformational shifts on the intracellular side. Together, our results provide important mechanistic insights into NaV channel slow inactivation.

Structural Advances in Voltage-Gated Sodium Channels.

Voltage-gated sodium (NaV) channels are responsible for the rapid rising-phase of action potentials in excitable cells. Over 1,000 mutations in NaV channels are associated with human diseases including epilepsy, periodic paralysis, arrhythmias and pain disorders. Natural toxins and clinically-used small-molecule drugs bind to NaV channels and modulate their functions. Recent advances from cryo-electron microscopy (cryo-EM) structures of NaV channels reveal invaluable insights into the architecture, activation, fast inactivation, electromechanical coupling, ligand modulation and pharmacology of eukaryotic NaV channels. These structural analyses not only demonstrate molecular mechanisms for NaV channel structure and function, but also provide atomic level templates for rational development of potential subtype-selective therapeutics. In this review, we summarize recent structural advances of eukaryotic NaV channels, highlighting the structural features of eukaryotic NaV channels as well as distinct modulation mechanisms by a wide range of modulators from natural toxins to synthetic small-molecules.

N-type fast inactivation of a eukaryotic voltage-gated sodium channel.

Voltage-gated sodium (NaV) channels initiate action potentials. Fast inactivation of NaV channels, mediated by an Ile-Phe-Met motif, is crucial for preventing hyperexcitability and regulating firing frequency. Here we present cryo-electron microscopy structure of NaVEh from the coccolithophore Emiliania huxleyi, which reveals an unexpected molecular gating mechanism for NaV channel fast inactivation independent of the Ile-Phe-Met motif. An N-terminal helix of NaVEh plugs into the open activation gate and blocks it. The binding pose of the helix is stabilized by multiple electrostatic interactions. Deletion of the helix or mutations blocking the electrostatic interactions completely abolished the fast inactivation. These strong interactions enable rapid inactivation, but also delay recovery from fast inactivation, which is ~160-fold slower than human NaV channels. Together, our results provide mechanistic insights into fast inactivation of NaVEh that fundamentally differs from the conventional local allosteric inhibition, revealing both surprising structural diversity and functional conservation of ion channel inactivation.