Molecular basis of the tarantula toxin jingzhaotoxin‐III (β‐TRTX‐Cj1α) interacting with voltage sensors in sodium channel subtype Nav1.5

Abstract

With conserved structural scaffold and divergent electrophysiological functions, animal toxins are considered powerful tools for investigating the basic structure-function relationship of voltage-gated sodium channels. Jingzhaotoxin-III (β-TRTX-Cj1α) is a unique sodium channel gating modifier from the tarantula Chilobrachys jingzhao, because the toxin can selectively inhibit the activation of cardiac sodium channel but not neuronal subtypes. However, the molecular basis of JZTX-III interaction with sodium channels remains unknown. In this study, we showed that JZTX-III was efficiently expressed by the secretory pathway in yeast. Alanine-scanning analysis indicated that 2 acidic residues (Asp1, Glu3) and an exposed hydrophobic patch, formed by 4 Trp residues (residues 8, 9, 28 and 30), play important roles in the binding of JZTX-III to Nav1.5. JZTX-III docked to the Nav1.5 DIIS3-S4 linker. Mutations S799A, R800A, and L804A could additively reduce toxin sensitivity of Nav1.5. We also demonstrated that the unique Arg800, not emerging in other sodium channel subtypes, is responsible for JZTX-III selectively interacting with Nav1.5. The reverse mutation D816R in Nav1.7 greatly increased the sensitivity of the neuronal subtype to JZTX-III. Conversely, the mutation R800D in Nav1.5 decreased JZTX-III's IC₅₀ by 72-fold. Therefore, our results indicated that JZTX-III is a site 4 toxin, but does not possess the same critical residues on sodium channels as other site 4 toxins. Our data also revealed the underlying mechanism for JZTX-III to be highly specific for the cardiac sodium channel.

Discovery of a selective Na V 1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models

“…Human Na V 1 α-subunits, the human Na V β1 subunit, and eGFP were transiently transfected into HEK293T cells and whole-cell patch-clamp recordings performed as previously described (33). The standard pipette solution contained: 140 mM CsF, 1 mM EGTA, 10 mM NaCl, 3 mM KCl, and 10 mM MgCl 2 , pH 7.3.…”

Discovery and characterization of NaV modulatory venom peptides

“…The model revealed key structural features, such as a hydrophobic face made up of five residues, W 7 , F 8 , Y 22 , W 30 and Y 32 . This structural feature is common with many Na V modulating, ICK motif, spider venom peptides [245] and has been hypothesized to contribute to the voltage sensor trapping mechanism of site 3 and site 4 gating modifier toxins [532]. The hydrophobic face has been theorized to allow the peptide to partition within the outer cell membrane, exposing a larger surface of interaction within the voltage sensor region [557].…”

“…However, some more recently discovered spider venom peptides have redefined the original role for site 4 modulation, which will be discussed later [243][244][245][246].…”

“…Multiple other spider venom peptides have since been discovered with critical binding domains located on the site 4 DIIS3-S4 loop [239] (Figure 4), as determined by either mutagenesis of key residues on this region of the Na V channel [243,245,255] or competitive assay with β-scorpion toxins [244]. However, many of these spider venom peptides exhibit a distinct functional profile to the β-scorpion toxins, whereby the peptide traps the DIIS4 voltage sensor in the closed state resulting in an inhibition of peak current [245,255,256].…”

“…However, many of these spider venom peptides exhibit a distinct functional profile to the β-scorpion toxins, whereby the peptide traps the DIIS4 voltage sensor in the closed state resulting in an inhibition of peak current [245,255,256]. Although a few of the jingzhaotoxins (Chilobrachys jingzhao) so far discovered have been shown to inhibit Na V current by acting as gating modifiers [244,[257][258][259], only one -β/κ-TRTX-Cj1a (jingzhaotoxin-III) -has so far demonstrated direct binding to the site 4 DIIS3-S4 loop [245]. Two other spider venom peptides that have been shown to bind the DIIS3-S4 linker through mutational analysis include µ-TRTX-Hh2a (huwentoxin-IV isolated from Ornithoctonus huwena) [243] and µ-TRTX-Hhn2a (hainantoxin-III isolated from Ornithoctonus hainana) [255].…”