In excitable cells, voltage-gated sodium (NaV) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance1,2. Inactivation is a hallmark of NaV channel function and is critical for control of membrane excitability3, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type NavAb channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of NavAb S6-cysteine mutants4, the pore-lining S6 helices and the intracellular activation gate have undergone significant rearrangements in which one pair of S6 segments has collapsed toward the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type NavAb models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains also shift around the perimeter of the pore module in NavAb, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in NaV channel gating and inactivation. These potential inactivated-state structures provide new insights into NaV channel gating and novel avenues to drug development and therapy for a range of debilitating NaV channelopathies.

Highlights d The structure of the cardiac sodium channel reveals key functional features d The antiarrhythmic drug flecainide blocks the pore below the selectivity filter d The ion selectivity filter and inactivation gate are revealed in atomic detail d An arrhythmia mutation creates a pathogenic gating pore 2 Å in diameter

Voltage-gated calcium (CaV) channels catalyze rapid, highly selective influx of Ca2+ into cells despite 70-fold higher extracellular concentration of Na+. How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial NaV channel NaVAb. Our results reveal interactions of hydrated Ca2+ with two high-affinity Ca2+-binding sites followed by a third lower-affinity site that would coordinate Ca2+ as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side-chains, which play a critical role in determining Ca2+ selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations, Cd2+ and Mn2+, with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca2+ ions bound in the selectivity filter and supports a “knock-off” mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our CaVAb model establishes a structural framework for understanding mechanisms of ion selectivity and conductance by vertebrate CaV channels.

Highlights d Voltage shifting and disulfide locking capture a resting-state structure of Na V Ab d Three gating charges translocate intracellularly through transmembrane electric field d Voltage sensor couples to pore opening by an elbow connecting S4 to the S4-S5 linker d Resting-state structure supports a sliding helix mechanism of gating