Molecular Basis Of Voltage-gated Sodium And Potassium Channel Interaction With Peptide Toxins From The Spider Chilobrachys Jingzhao Venom

Spider venom is an important source of peptide molecules with different pharmacological properties. With high binding affinity and diverse pharmacological functions, peptide toxins are powerful ligands to investigate the structure-function relationships of voltage-gated ion channels. Jingzhaotoxin-Ⅲ (β-TRTX-Cj1α) is a36-residue peptide from the tarantula Chilobrachys jingzhao venom. The toxin is specific for Navl.5and Kv2.1channels over the majority of other ion channel subtypes. In order to better understand the molecular basis of JZTX-Ⅲ interaction with Kv2.1, we investigated the bioactive surface of JZTX-Ⅲ and the toxin binding site on Kv2.1. The results indicated that JZTX-Ⅲ docked onto the Kv2.1voltage sensor paddle. Alanine replacement of each residue Phe274, Lys280, Ser281, Leu283, Gln284, or Val288could decrease JZTX-Ⅲ affinity by>7-fold. Among them, Ser281is the most crucial determinant, and the substitution with Ala, Phe, lie, Val, or Glu increased the IC50value by>34-fold. The bioactive surface of JZTX-Ⅲ interacting with Kv2.1is composed of four hydrophobic residues (Trp8, Trp28, Trp30, and Val33) and three charged residues (Arg13, Lys15, and Glu34). Although a hydrophobic patch, mainly composed of Trp8, Trp28, and Trp30, was shared in the interaction with Nav1.5and Kv2.1, the bioactive surfaces of JZTX-Ⅲ interacting with both channels were not identical. Residues Asp1, Glu3, and Trp9were critical only for the inhibition of Nav1.5, whereas three charged residues (Arg13, Lys15, and Glu34) and one hydrophobic residue (Va133) were crucially involved in the interaction with Kv2.1. The bioactive surfaces of JZTX-Ⅲ interacting with Nav1.5and Kv2.1are only partially overlapping, but it inhibits these two distinct types of ion channels perhaps by recognizing a conserved binding motif on the voltage sensor paddle. Taken together, our study confirms special molecular mechanisms responsible for JZTX-Ⅲ binding to Nav and Kv channels.To better understand the structure-function relationships, we analyzed the effects of JZTX-Ⅲ on rNav1.8currents expressed in ND7/23cells. Our finding showed that JZTX-Ⅲ could inhibit the activation of rNav1.8channel. Based on the high sequence similarity in the DIIS3-S4linkers of hNavl.5and rNavl.8, we propose that JZTX-Ⅲ inhibits rNavl.8channel perharps by binding to the receptor site4. JZTX-Ⅲ might be a peptide toxin with potential to shed light on the molecular mechanism of Nav1.8blocking.Jingzhaotoxin-Ⅰ,-Ⅸ, and-Ⅺ (JZTX-Ⅰ,-Ⅸ, and-Ⅺ) are important neurotoxins from the tarantula Chilobrachys jingzhao venom. They have three disulfide bonds with the linkage of Ⅰ-Ⅳ, Ⅱ-Ⅴ, and Ⅲ-Ⅵ that is a typical pattern found in inhibitor cystine knot molecules. JZTX-Ⅰ is composed of33residues and exhibits limited sequence similarity with any reported peptide but nearly50%with JZTX-Ⅲ and GxTX1E. JZTX-Ⅸ has35amino acid residues and shares high sequence similarity with other peptides from the tarantula venoms such as HaTx1(63%), HmTX-Ⅰ (63%), SGTx1(60%), and JZTX-Ⅺ (71%). JZTX-Ⅰ,-Ⅸ, and-Ⅺ as gating modifiers are able to inhibit the activation of the potassium channel subtype Kv2.1and reduce currents through Kv2.1channels by modifying channel gating. They could significantly accelerate the kinetic of deactivation of Kv2.1channels, which reflects the transition from the activated state to the closed state, suggesting that these toxins might alter Kv2.1activation by docking onto the voltage sensor paddles. The inhibitions were concentration-dependent with the IC50values of8.05,1.35and0.57μM, respectively. In order to investigate their binding receptor sites on the Kv2.1channel, individual amino acid residue in the S1-S2linker and the S3b-S4paddle motif was mutated to alanine. Site-directed mutagenesis analysis demonstrated that four residues (Ile273, Phe274, Glu277, and Lys280) in the S3b-S4motif contributed to the formation of JZTX-I binding receptor site. The mutations I273A, F274A, E277A, and K280A reduced toxin binding affinity by6-,10-,8-, and7-fold, respectively. In addition, three residues (Ile273, Phe274, and Glu277) in the S3b region greatly reduced the binding affinity of both JZTX-Ⅸ and JZTX-Ⅸ for Kv2.1channel. The mutations I273A, F274A and E277A decreased JZTX-Ⅸ sensitivity by9-,19-,16-fold and JZTX-Ⅺ sensitivity by7-,11-,13-fold, respectively. Taken together with the findings that these toxins accelerate channel deactivation, these results suggest that they all inhibit Kv2.1activation by docking onto the voltage sensor paddles and trapping the voltage sensors in the closed state.Our previous work have shown that JZTX-Ⅰ is an α-Like toxin that could inhibit channel fast inactivation kinetics of TTX-R sodium channels on rat cardiac myocytes and TTX-S sodium channels expressed on rat DRG neurons as well as cotton boll worm central nerve ganglian neurons. Using whole-cell patch-clamp technique, we analyzed the actions of JZTX-Ⅰ on voltage-gated sodium channel subtypes. Our findings indicated that JZTX-Ⅰ could inhibit Nav1.5channel with high affinity (IC50=0.33μM). The inhibition by JZTX-Ⅰ was in a time-dependent manner, and it had no effect on activation kinetics and steady-state inactivation. Alanine-scanning mutagenesis of Navl.5DIV S3-S4extracellular linker indicated that residue D1609was a critical molecular determinant for JZTX-Ⅰ binding. These results suggest that JZTX-Ⅰ is a site3toxin that inhibits channel inactivation by docking at the extracellular S3-S4linker of domain Ⅳ.