Abstract
Voltage-gated sodium channels (VGSCs; NaV1.1–NaV1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7–2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.
Highlights
Voltage-gated sodium channels (VGSCs), the current of which was discovered by Nobel laureatesHodgkin and Huxley in 1952, are critical elements of cellular function because they participate in the generation and propagation of action potentials in excitable cells, such as muscle cells and neuron [1,2,3,4,5].The crystal structure of the homotetrameric bacterial sodium (Na) channel NaVAb was solved by Payandeh et al at high resolution (2.7 Å) in 2011 and provided insights into the atomic structure of mammalian sodium channels at some level [6]
We investigated the toxin’s selectivity for four human (h) or rat (r) VGSC subtypes and the specific sites involved in the action of JZTX-V on NaV1.4
We observed that 100 nM JZTX-V could rapidly and completely inhibit wild-type rNaV1.4 (n = 6), whereas treatment with 500 nM JZTX-V decreased activation of wild-type hNaV1.5 by only 43% ±6.5%
Summary
Voltage-gated sodium channels (VGSCs), the current of which was discovered by Nobel laureates. Toxins from various organisms have been used to describe eight different receptor sites on the α subunits of VGSCs, all of which are linked to specific effects on channel function [27] For most of these toxins, the precise pattern of subtype selectivity is either unknown or, at best, fragmentary. NaSpTx families 1–12, related spider venom peptides that act on VGSCs, have been well-defined recently in terms of their activities and sequence similarities. Among these 12 families, NaSpTx family 3 has 14 members that are 29–32 peptides long, with highly conserved sequences in the. JZTX-V has a much stronger effect on NaV1.4 over other tested subtypes, and the bioactive face of this toxin is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20, K22) residues
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