Screening Spider Toxin Libraries for TRP Channel Inhibitors

Abstract

Pain caused by activation of pain-sensing peripheral neurons (“nociceptors”) is a major source of human suffering and economic loss. A variety of stimulus- and voltage-gated ion channels in peripheral nociceptors play essential roles in the transduction and transmission of normal and pathological pain signals in human patients, and thus are important targets for pharmacological intervention. Around one hundred inhibitory cystine knot (ICK) peptide toxins were selected from a database of published and unpublished spider toxins. The tethered-toxin (“t-toxin”) method for the heterologous expression of ICK ion channel toxins in membrane tethered form directly in a channel-expressing target cell was applied. Pools of cRNA of t-toxins (∼6 per pool) were co-expressed with ion channels in Xenopus oocytes and the currents were measured by two-electrode voltage clamp 2-4 days after injection. One pool was found to cause ∼50 % suppression of the TRPA1 current induced by 100 μM mustard oil, and subscreening of this pool revealed a single t-toxin responsible for this effect. When co-expressed with TRPV1, this t-toxin suppressed the inward current induced by 20 μM capsaicin ∼80%. The effect of this t-toxin on other ion channels was also investigated. It completely blocked depolarization-activated inward currents of Para, a Drosophila voltage-gated sodium channel. When co-expressed with Nav1.2, it reduced peak currents by 60%. Interestingly, however, it did not affect currents flowing through Kir4.1, an inwardly rectifying potassium channel. These results suggest that this relatively promiscuous toxin acts on a structural feature common to ion channels with six transmembrane domains (Trp channels and voltage-gated channels), but lacking in those that only possess the two pore-spanning transmembrane domains (inward rectifier).