Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7. Issue 3 (19th March 2020)

• Record Type: Journal Article
• Authors: Tzakoniati, Foteini; Xu, Hui; Li, Tianbo; Garcia, Natalie; Kugel, Christine; Payandeh, Jian; Koth, Christopher M.; Tate, Edward W.
• Is Part Of: Cell chemical biology. Volume 27:Issue 3(2020)
• Page Start: 306
• Page End: 313.e4
• Publication Date: 2020-03-19
• Subjects: Nav1.7 -- Huwentoxin-IV -- photoaffinity labeling -- diazirine -- peptide mapping -- photocrosslinking -- voltage-gated sodium channel -- inhibitory cystine knot; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2019.10.011
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: Voltage-gated sodium (Nav) channels respond to changes in the membrane potential of excitable cells through the concerted action of four voltage-sensor domains (VSDs). Subtype Nav1.7 plays an important role in the propagation of signals in pain-sensing neurons and is a target for the clinical development of novel analgesics. Certain inhibitory cystine knot (ICK) peptides produced by venomous animals potently modulate Nav1.7; however, the molecular mechanisms underlying their selective binding and activity remain elusive. This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of Nav1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-S2 and helix S3 of VSD2 in a chimeric channel system. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on Nav channels. Graphical Abstract: Highlights: Development of six potent diazirine-containing photoprobes based on Huwentoxin-IV Photoprobes specifically photolabel purified bacterial-Nav1.7 VSD2 chimeric channels Proteomic mass spectrometry identifies binding site on S1-S2 loop and S3 helix Proposed model of HwTx-IV binding reveals importance of K27 and R29 Abstract : Classical structural studies of toxin and ion channel complexes are very challenging, and not always feasible. Tzakoniati et al. used photocrosslinking and mass spectrometry to validate and provide direct evidence for specific binding interactions between a toxin peptide and the voltage-gated sodium channel Nav1.7.