Molecular interactions of the gating modifier toxin ProTx-II with Na _v1.5: Implied existence of a novel toxin binding site coupled to activation

Voltage-gated Na⁺ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na_v and Ca_v, but not K_v, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na_v1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K_v channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na_v1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na _v channels with potential to shed new light on this process.