Identification of new batrachotoxin-sensing residues in segment IIIS6 of the sodium channel

Abstract

Ion permeation through voltage-gated sodium channels is modulated by various drugs and toxins. The atomistic mechanisms of action of many toxins are poorly understood. A steroidal alkaloid batrachotoxin (BTX) causes persistent channel activation by inhibiting inactivation and shifting the voltage dependence of activation to more negative potentials. Traditionally, BTX is considered to bind at the channel-lipid interface and allosterically modulate the ion permeation. However, amino acid residues critical for BTX action are found in the inner helices of all four repeats, suggesting that BTX binds in the pore. In the octapeptide segment IFGSFFTL in IIIS6 of a cockroach sodium channel BgNa(V), besides Ser_3i15 and Leu_3i19, which correspond to known BTX-sensing residues of mammalian sodium channels, we found that Gly_3i14 and Phe_3i16 are critical for BTX action. Using these data along with published data as distance constraints, we docked BTX in the Kv1.2-based homology model of the open BgNa(V) channel. We arrived at a model in which BTX adopts a horseshoe conformation with the horseshoe plane normal to the pore axis. The BTX ammonium group is engaged in cation-π interactions with Phe_3i16 and BTX moieties interact with known BTX-sensing residues in all four repeats. Oxygen atoms at the horseshoe inner surface constitute a transient binding site for permeating cations, whereas the bulky BTX molecule would resist the pore closure, thus causing persistent channel activation. Our study reinforces the concept that steroidal sodium channel agonists bind in the inner pore of sodium channels and elaborates the atomistic mechanism of BTX action.

FIGURE 1

Residues Gly³ⁱ¹⁴ and Phe³ⁱ¹⁶ in IIIS6 are critical for the action of BTX.A, sodium currents before and after the application of 500 nm BTX. The BTX-induced noninactivating current and tail current were elicited by a 20-ms test pulse to −10 mV from a holding potential of −120 mV after 3000 repetitive pulses to −10 mV at a frequency of 10 Hz in the presence of 500 nm BTX. B and C, voltage dependence of activation (B) and inactivation (C). Data from a previous study (10) and the current study were pooled to generate activation and inactivation curves in B and C. D and E, effects of amino acid substitutions on BTX-induced tail current (D) and noninactivating current (E). The amplitude of tail current and noninactivating current induced by BTX was normalized to the peak current after toxin application. F–H, voltage dependence of inactivation of G³ⁱ¹⁴A (F), F³ⁱ¹⁶A (G), and F³ⁱ¹⁶K (H) channels before and after the application of BTX. I, percentage of channels modified by BTX for BgNa_v1-1a and mutants. The voltage dependence of activation (conductance curves) in the presence of BTX was fitted with the sum of two Boltzmann relationships to determine the percentage of channels that were modified by BTX. An asterisk indicates a significant difference from the wild-type channel as determined by t tests (p < 0.05).

FIGURE 2

Structural formulae of BTX.

FIGURE 3

Predicted binding mode of BTX in BgNa_v1-1. The pore-forming domain of the channels is shown with the inner helices (thick rods), outer helices (thin rods), P-helices (ribbons), and ascending limbs (thin rods). Repeats I, II, III, and IV are colored orange, cyan, green, and blue, respectively. BTX-sensing residues are space-filled. BTX is shown by sticks with yellow carbons, red oxygens, and blue nitrogens. BTX adopts a horseshoe conformation with its exterior predominantly hydrophobic side interacting with the inner helices and interior hydrophilic side exposed to the pore axis. A and B, top views. C and D, side views with the front repeat removed for clarity. E, schematic view of BTX-channel interactions.

FIGURE 4

BTX in the open state (A, C, and E) and closed state (B, D, and F) models of the sodium channel. For clarity, only parts of the outer and inner helices around level i15 are shown at the top (A–D) and side (E and F) views. The front helix IS6 is removed at E (except for BTX-sensing residue Ser¹ⁱ¹⁵), and repeats I and IV are removed at F. In the open state, the central inner pore cavity at level i15 is wide enough to accommodate BTX in the horseshoe conformation. The van der Waals shape of the BTX horseshoe conformation approximately fits the central inner cavity (C). An Na⁺ ion (orange sphere) binds to the inner surface of the BTX horseshoe (A and C). B, KcsA-based model of the closed channel with BTX constrained at the same level as in A. Due to the small lumen of the closed pore, this orientation of BTX is unstable. D and F, upon removal of the BTX-channel distance constraints, BTX extend along the pore axis of the closed pore. This model shows how BTX could be trapped in the closed channel.

FIGURE 5

BTX sensitivity of seven mutants generated to test the horseshoe model of BTX binding.A and B, effects of amino acid substitutions on BTX-induced tail current (A) and noninactivating current (B). C, percentage of channels modified by BTX. The recording protocols and data analysis are the same as those described in the legend to Fig. 1. The data are presented as mean ± S.D. An asterisk indicates a significant difference from the wild-type channel as determined by t tests (p < 0.05).