Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

Nav1.x, eukaryotic voltage-gated sodium channels (in mammals, x = 1-9, where numbers denote different isoforms, associated primarily with a specific tissue or cell type) VGIC, voltage-gated ion channels, whose subfamilies include Cav, Kv, and Nav variants, based on their ion selectivity, and in a broader sense, any ion channel controlled in part by a voltage-sensing domain of four transmembrane segments, with the basic residues that sense the transmembrane electric field, concentrated in S4

Nav conformational states
Resting: Nonconducting states favored by negative or hyperpolarized voltages across cell membranes.
Open: Activated, conducting state normally transiently favored by membrane depolarization.
Inactivated: Nonconducting states typically favored by prolonged membrane depolarization.
Nav steady-state and kinetic parameters τ deact , deactivation time constant I tail , current amplitude immediately after repolarization to V h I tailmax , maximal current amplitude immediately after repolarization to V h SPI, the empirical description of the inactivation seen as a decay of current/conductance during a prolonged depolarizing pulse and does not necessarily imply a specific mechanism or a single inactivated state SSI, steady-state inactivation τ inact , time constant of current decay during an activating pulse I Px , peak current at pulse x τ rise , time constant of early current development upon depolarization I peak , maximal current amplitude during an activating pulse Figure S1. The reduction of peak current amplitude with BTX modification is not associated with cumulative inactivation. (A) Diary plot of NaChBac (NB) relative peak current during BTX modification at 1.0 Hz (n = 5). (B) Diary plot of NB relative peak current during 0.1 Hz (n = 4) and 1.0 Hz (n = 4) stimulation in control conditions. V h = −120 mV; V t = −10 mV; 25-ms pulses. Figure S2. Cumulative inactivation of NaChBac currents at different holding potentials. Diary plot of NaChBac (NB) relative peak currents in control (black) and after BTX (red) modification, displayed for 300-ms pulses to −10 mV; 0.1 Hz; V h = −120 mV (circles), −100 mV (triangles), and −80 mV (squares; n = 4 per condition). Figure S3. Selectivity, calculated as relative permeability, P K /P Na , from the reversal potential. (A) Whole-cell NaChBac currents in response to 500ms activating pulses with 10 µM BTX in the pipette. Left: Symmetrical Na + (Na i /Na o , black). Right: Na i /K o (red). I-V is from −120 mV to 100 mV; V h = −120 mV. (B) Peak I-V relationships for currents from A. Based on the shift of reversal potential, P K /P Na = 0.04 is approximately twofold higher than previously determined for unmodified NaChBac channels. Figure S4. NaChBac mutations (N225 i20 K and N225 i20 A) render the channel nonfunctional. These mutations are homologous to the rNav1.4 domain 1 mutants N434 i20 K and N434 i20 A (see Fig. 5). Note that the homologous mutation in eukaryotic Nav channels replaces one amino acid change in a single domain and reduces the ability of BTX to modify the channel. In an assembled homotetrameric bacterial channel, this single mutation would result in four single-residue substitutions, one in each of the four subunits.

Finol-Urdaneta et al.
Batrachotoxin activation of NaChBac and NavSp1 S18 a Prefixes 1, 2, 3, and 4 designate NaChBac subunits that correspond to repeat I, II, II, and IV, which in an extracellular view of the eukaryotic sodium channel are arranged clockwise (Dudley et al., 2000). b Na I and Na III designate positions of sodium ions in the NavMs x-ray structure (Sula et al., 2017). Na IV designates putative position of sodium ion at the focus of P1 helices (Tikhonov and Zhorov, 2017). c Contributions with the absolute energy value <0.2 kcal/mol are not shown In general, the time-dependent forward (α) and backward (β) transition rates are given by where V(t) is the applied voltage at time t, T is temperature, k b is Boltzmann's constant, α o and β o are the rates at zero applied voltage, and q is the charge moved within the membrane electric field up to the transition state intermediate. Note that we have made the simplifying assumption that the magnitude of q is the same for both forward and backward rates, which in general is not an absolute requirement. See also the kinetic modeling section in Materials and methods. In the simulations shown in Fig. 8, the following restrictions were applied. For control traces, the α3/β3 charges were held at zero, and the rate constants α4/β4, and their associated charges were set to zero. For BTX traces, all charges shown with a zero value were held at zero, while all other charges were allowed to vary.