Abstract
SummaryVoltage-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.
Highlights
Voltage-gated sodium (Nav) channels initiate and propagate action potentials in excitable cells by permitting the inward flux of Na+ ions in response to changes in membrane potential
We present the development of a library of potent photocrosslinking probes of HwTx-IV and demonstrate specific photocrosslinking to a purified Nav1.7 VSD2-NavAb chimeric system by SDS-PAGE shift assays and tandem mass spectrometry (MS) (MS2)
Development of Potent HwTx-IV Toxin Photoprobes Targeting Nav1.7 HwTx-IV is a 35-amino acid, C-terminally amidated inhibitory cystine knot (ICK) peptide with three conserved disulfide bridges (C1-C4, C2-C5, and C3-C6) that stabilize a knotted structure (Figure 1A)
Summary
Voltage-gated sodium (Nav) channels initiate and propagate action potentials in excitable cells by permitting the inward flux of Na+ ions in response to changes in membrane potential (reviewed in Catterall, 2000). Gain-of-function mutations cause painful phenotypes such as inherited erythromelalgia and paroxysmal extreme pain disorder (Dib-Hajj et al, 2013), whereas loss-of-function mutations lead to an inability to sense pain, and anosmia, in otherwise healthy individuals (Cox et al, 2006). This genetic evidence highlights the direct involvement of Nav1.7 in pain signaling, and has stimulated intense efforts to discover and develop novel analgesics targeting Nav1.7 that, if sufficiently specific, would presumably not exhibit many of the unfavorable liabilities of current treatments such as those associated with opioids
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