A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel

Abstract

Voltage-gated sodium channels (Na V s) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial Na V s have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial Na V s. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic Na V s nonselective voltage-gated, Bacillus alcalophilus (Ns V Ba) and bacterial sodium channel from Bacillus halodurans (NaChBac) (IC 50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of Ns V Ba, whereas the local anesthetic drug lidocaine was shown to antagonize Ns V Ba without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of Ns V Ba, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of Ns V Ba in one of the deactivated states. In mammalian Na V s, JZTx-27 preferably inhibited the inactivation of Na V 1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic Na V s. More important, we proposed that JZTx-27 stabilized the Ns V Ba VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of Na V s.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V. DeCaen, P. G. Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel. FASEB J. 31, 000-000 (2017). www.fasebj.orgMutations in Na V s cause a variety of diseases of the heart and central and peripheral nervous systems (e.g., long QT syndrome and epilepsy) (1, 2). Many of these mutations are found within the voltage-sensor modules (VSMs) of Na V s, which alter the voltagedependent kinetics of the channel gating (3-5). Understanding structure and function of the voltage sensor of Na V s will provide insight into the molecular basis of electrical signaling in normal and diseased conditions. Eukaryotic Na V s are large proteins with 24 transmembrane segments, making them challenging to study by using crystallographic techniques (6). Several laboratories have crystalized Na V s from bacteria, which are relatively small and homotetrameric (7-10). Four monomers of bacterial Na V s assemble to form a channel. Analogous to the 4 domains (I-IV) of eukaryotic Na V s, each bacterial channel monomer contains a voltage sensor and a pore domain. For prokaryotic Na V s, the first 4 transmembrane segments (S1-4) form a voltage sensor module (VSM) and the last 2 transmembrane segments (S5 and -6) compr...

Purification and Characterization of JZTx-14, a Potent Antagonist of Mammalian and Prokaryotic Voltage-Gated Sodium Channels

“…The mutation study showed that JZTx-14 acted on NaChBac by binding to the S3–4 extracellular loop, with the 108th phenylalanine (F108) being the key residue. This action mode resembled the previously reported NaChBac channel toxin JZTx-27, although their key residues in the channel were different [ 29 ]. We speculate that JZTx-14 trapped the NaChBac voltage sensor in the deactivated state as it positively shifted the activation kinetics.…”

“…It was also active on bacterial Na V s NaChBac and NsvBa, as revealed by our previous screening analysis of spider venom peptides for bacterial Na V s antagonists. However, the toxin was not deeply explored as a bacterial Na V toxin, as it was approximately 10 folds less potent than the toxin JZTx-27, which was investigated in our previous study [ 29 ]. This toxin was purified to homogeneity ( Figure 1 B,C), and its molecular weight was determined as 3422.17 Da (M + H + ) ( Figure 1 C).…”

“…For the bacterial Na V s NaVPz and NaVSP, 1 µM JZTx-14 only caused a 34.5 ± 6.5% and 25.7 ± 5.7% inhibition of their currents, respectively ( Figure 4 D,E, n = 3). JZTx-27 is another NaChBac and NsvBa channel antagonist characterized in the same venom [ 29 ]. In the RP-HPLC purification of the venom, the JZTx-27 peak followed those of JZTx-14 and JZTx-2 ( Figure 1 A; the arrows labeled peaks).…”

“…Our previous study showed that JZTx-27 bound with the S3–4 extracellular loop of NaChBac, with the 103th phenylalanine (F103) being the key residue [ 29 ]. We analyzed the key residues in NaChBac for binding JZTx-14 in the S3–4 loop region by using an alanine scan strategy.…”

Scorpion toxin inhibits the voltage‐gated proton channel using a Zn2+‐like long‐range conformational coupling mechanism

“…We compared the mechanism of AGAP/W38F inhibiting H V 1 channel with that of Zn 2+ , as well as that of some other gating modifier toxins inhibiting Na V and K V channels. Our electrophysiological data suggest that AGAP/W38F inhibition of H V 1 channel shared a canonical voltage‐sensor trapping mechanism with gating modifier toxins inhibiting Na V and K V channels (Lee, Wang, & Swartz, ; Tang et al, ; Xiao et al, ). AGAP/W38F and Zn 2+ act on H V 1 channel with certain similarities.…”