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.

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Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltagegated sodium channel (Na V Sp1) PD forms a stand-alone, ion selective pore (Na V Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na V Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na V Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca V Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na V s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na V Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na V Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.V oltage-gated sodium channels (Na V s) are large polytopic membrane proteins involved in action potential generation in excitable cells and belong to an ion channel superfamily that includes voltage-gated calcium channels (Ca V s), voltage-gated potassium channels (K V s), and transient receptor potential (TRP) channels (1, 2). Within the voltage-gated ion channel (VGIC) superfamily, Na V s and Ca V s are close relatives (1-3) that share a topology of 24 transmembrane segments organized in four homologous six-transmembrane repeats. These two families are also thought to share some common structure in the ion selectivity filter despite having markedly different ion permeation properties (4). Both are central to human neuromuscular, cardiovascular, and neural physiology. Consequently, they are targets for a host of pharmaceuticals used to treat a diverse set of disorders and remain active targets for drug development (5-7). Recently, single subunit, six-transmembrane segment Na V s have been identified in a large number of bacteria from diverse environments (8, 9). These channels show clear similarities to eukaryotic Na V s and Ca V s (2, 9, 10), suggesting that the prokaryotic channels may have been ancestors of the more complex vertebrate channels.Despite the central importance of Na V s, nothing is known about the high-resolution structure o...

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