NaV channels are critical for the upstroke of the action potential in peripheral sensory neurons. These neuron express a unique subset of NaV channels including NaV1.7, NaV1.8 and NaV1.9. Mutations in these isoforms have been associated with both painful and painless neuropathies. Potent and selective pharmacological probes of NaV channels are required to further understand the role of particular NaV channels in pain sensing. However this endeavour is hampered by the high sequence homology (>50%) between isoforms. The -scorpion toxin Tf2 was previously identified as a selective agonist of NaV1.3. The role of NaV1.3 in PNS is not well defined with low levels reported in adult rodents PNS but upregulation reported after injury. Tf2 and analogs were generated using native chemical ligation, assessed using high-throughput and high content fluorescence imaging, manual and automated patch clamp and in vivo behavioural nociception experiments. We confirm the -scorpion toxin action at NaV1.3 including a strong hyperpolarizing shift in activation (~20 mV). Tf2 does not activate NaV1.1,1.2-1.7 in high-throughput fluorescent assay. Tf2 does not shift the voltage-dependence of activation of NaV1.8. Interestingly, Tf2 induced a small hyperpolarizing shift in activation of NaV1.9. Tf2 induced calcium influx in DRG neurons of all sizes. When injected intraplantar Tf2 caused spontaneous nocifensive behaviors in WT and NaV1.9-/- animals the absence of changes in withdrawal to mechanical or heat stimulus. Therefore, Tf2 induces spontaneous nocifensive behaviours when injected intraplantar not caused by NaV1.9 activation alone.

SELECTIVE PEPTIDE TOXINS FOR ASSESSING NAV CHANNEL FUNCTION IN SENSORY NEURONS