The
voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory
pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus)
venom proteins inhibit Nav1.8 and block
pain in grasshopper mice (Onychomys torridus). These
proteins provide tools for examining Nav1.8 structure-activity relationships. To identify
proteins that inhibit Nav1.8 activity,
venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique
peptides from inhibitory subfractions. A search of the
peptides against the AZ bark
scorpion venom gland transcriptome revealed four novel
proteins between 40 and 60% conserved with
venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82
amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (
tryptophan,
tyrosine, or
phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that
proteins may function as gating modifiers as opposed to pore blockers.