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.

Brain I A and cardiac I to currents arise from complexes containing Kv4 voltage-gated potassium channels and cytoplasmic calcium-sensor proteins (KChIPs). Here, we present X-ray crystallographic and small-angle X-ray scattering data that show that the KChIP1-Kv4.3 Nterminal cytoplasmic domain complex is a cross-shaped octamer bearing two principal interaction sites. Site 1 comprises interactions between a unique Kv4 channel N-terminal hydrophobic segment and a hydrophobic pocket formed by displacement of the KChIP H10 helix. Site 2 comprises interactions between a T1 assembly domain loop and the KChIP H2 helix. Functional and biochemical studies indicate that site 1 influences channel trafficking, whereas site 2 affects channel gating, and that calcium binding is intimately linked to KChIP folding and complex formation. Together, the data resolve how Kv4 channels and KChIPs interact and provide a framework for understanding how KChIPs modulate Kv4 function.In biological systems, electrical information is encoded and processed by changes in actionpotential timing, duration, frequency, waveform and number 1 . Modulation of voltage-gated potassium channels is central to these events and affects heart rate, sensory transduction and cognition. The ion transport 2 and voltage-dependent gating properties 3 of the potassium channel α subunits that form the ion conduction pathway are well characterized. Although α subunits form the pore, many channels function as complexes that require cytoplasmic and transmembrane auxiliary subunits 4 . To date, little is known regarding the way in which such components bind α subunits and modulate channel action. Understanding the interplay between regulatory components and pore-forming domains is crucial for unraveling how modulatory signals 4 and homeostatic mechanisms 5 allow excitable cells to sense and respond to environmental cues. Two potassium currents, I A (ref. 6 ) and I to (ref. 7 ), exert strong control over neuronal and cardiac excitability, respectively, and provide clear examples of the importance of auxiliary subunits for tuning properties of α subunits. Both currents arise from complexes of Kv4 The structure of the KChIP1-Kv4.3 T1 complex shows that the assembly forms a crossshaped octamer having the T1 tetramer at the center (Fig. 1a, chains A-D) and individual KChIPs extending radially (Fig. 1a, chains E-H). The asymmetric unit has two octameric complexes that are apposed on the cytoplasmic T1 faces (Fig. 1b). T1 shows few differences from the isolated Kv4.3 T1 structure 17 (r.m.s. deviation = 0.64 Å 2 over 408 Cα positions). In contrast, KChIP1 shows substantial differences (r.m.s. deviation = 4.03 Å 2 over 179 Cα positions) from isolated KChIP1 (ref. 17 ).The structure reveals two main interaction sites. Site 1 buries ~2,100 Å 2 total surface area between residues 3 and 21 of a conserved hydrophobic segment that is on the N-terminal side of T1 (called T1N; Fig. 2a) and a large hydrophobic pocket (28 Å long, 12 Å deep, 10 Å wide) formed by KChIP1 (Fig. 2b,c...

Class III adenylyl cyclases contain catalytic and regulatory domains, yet structural insight into their interactions is missing. We show that the mycobacterial adenylyl cyclase Rv1264 is rendered a pH sensor by its N-terminal domain. In the structure of the inhibited state, catalytic and regulatory domains share a large interface involving catalytic residues. In the structure of the active state, the two catalytic domains rotate by 55 degrees to form two catalytic sites at their interface. Two alpha helices serve as molecular switches. Mutagenesis is consistent with a regulatory role of the structural transition, and we suggest that the transition is regulated by pH.

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 (BacNaV) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNaV 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 BacNaV 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 BacNaV voltage dependencies, and demonstrate that a discrete domain can encode the temperature dependent response of a channel.