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-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.Voltage-gated sodium channels (Na V ) are responsible for the rapid rising phase of the action potential in nerve and muscle cells. The pore-forming ␣-subunit of Na V channels contains four repeats, and each repeat has six transmembrane helices (S1-S6). The S1-S4 helices form the voltage sensor domain.Positively charged S4 helices move outward in response to membrane depolarization. The S5 and S6 helices contribute to the pore-forming domain. The extracellular linkers connecting S5 and S6 helices form the four re-entrant P-loops, which contain the selectivity filter residues. In the absence of x-ray structures of Na V channels, their homology models, which are based on x-ray structures of potassium channels, are used to explain structure-activity relationships of various sodium channel ligands, including local anesthetics (1-4), steroidal activators (5-8), and pyrethroid insecticides (9, 10). Recent reinterpretation of data on substituted cysteine accessibility of the Ca V 2.1 channel (11) in view of a the channel homology model, which is based on the x-ray structure of the open voltage-gated potassium channel Kv1.2 (12), further supports a general similarity of the inner pore architecture in different voltage-gated cationic channels (13).Sodium channels are targets for numerous drugs and naturally occurring toxins. Batrachotoxin (BTX) 4 is a steroidal sodium-channel agonist, which...

Constraint shapes convergence in tetrodotoxin-resistant sodium channels of snakes

“…However, these toxins do not bind to the outer pore but instead to the inner pore (BTX) (48)(49)(50) or to other transmembrane helices (PTX-B) (47,51). Thus, although overall Na + channel structure in L. epinephelus likely is influenced by multiple prey toxins, we are confident that the P-loop replacements we discuss have been shaped by selection from TTX, because the outer pore is the target of TTX, and these functional mutations are at sites critical to TTX binding but uninvolved in BTX or PTX-B binding (47)(48)(49)(50)(51).…”

Batrachotoxin, Pyrethroids, and BTG 502 Share Overlapping Binding Sites on Insect Sodium Channels

“…Mutational studies show that BTX binds in the inner pore, contacts the inner helices from all four repeats (Wang et al, 2006(Wang et al, , 2007a, and may adopt an ion-permeable "horseshoe" conformation within the channel as we recently proposed (Du et al, 2011a). In contrast, pyrethroids including deltamethrin are proposed to bind in the lipid-exposed interface between the linker-helix IIS4-S5, the outer helix IIS5, and the inner helix IIIS6 (O'Reilly et al, 2006;Du et al, 2009).…”

“…2A; Supplemental Table S2), that are critical for the action of pyrethroids (Du et al, 2009). In addition, we have shown that Gly 3i14 and Phe 3i16 are also critical for the action of BTX (Du et al, 2011a). To determine whether these residues are also critical for the action of BTG 502, we examined the effect of BTG 502 on these four mutant channels.…”

“…However, mutational studies identified BTX-sensing residues in the inner helices of all four domains, suggesting that BTX is directly exposed to the permeation pathway (Tikhonov and Zhorov, 2005b). More recent mutational and modeling studies have confirmed this prediction (Wang et al, 2006(Wang et al, , 2007aDu et al, 2011a). Studies of the mechanisms of insect resistance to pyrethroids led to the identification of pyrethroid-sensing residues in diverse regions of insect sodium channels (Soderlund, 2005;Davies et al, 2007;Dong, 2007).…”

“…These data have been used to construct a model of the sodium channel in which pyrethroids bind to a cavity formed by the linker-helix IIS4 -S5, the outer helix IIS5, and the inner helix IIIS6 at the interface between domains II and III (O'Reilly et al, 2006). Systematic site-directed mutagenesis of residues in IIIS6 revealed additional four residues that are important for the action of deltamethrin (Du et al, 2009), and two of them are also critical for the action of BTX (Du et al, 2011a). Despite this apparent overlap, the binding sites for BTX and pyrethroids in IIIS6 are distinct, involving opposite faces of the IIIS6 transmembrane helix (Du et al, 2009).…”