Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. Here, we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNa_V) 'pore only' proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained. This context-independent structural robustness, supported by molecular dynamics simulations, indicates that VGIC-PD tertiary structure is independent of quaternary interactions. This fold occurs throughout the VGIC superfamily and in diverse transmembrane and soluble proteins. Strikingly, characterization of PD subunit-binding Fabs indicates that non-canonical quaternary PD conformations can occur in full-length VGICs. Together, our data demonstrate that the VGIC-PD is an autonomously folded unit. This property has implications for VGIC biogenesis, understanding functional states, de novo channel design, and VGIC structural origins.