Probing TRPV1 Structure with Limited Proteolysis

Abstract

TRPV1 is a tetrameric voltage-sensitive cation channel that is activated by heat and vanilloids. The architecture of TRP channels are thought to be related to Kv channels, with each subunit containing six transmembrane segments. In addition to being activated by capsaicin, TRPV1 is activated by Double-Knot Toxin (DkTx), a protein toxin purified from the Chinese Bird Spider, Selenocosmia huwena (Bohlen et al. 2010, Cell 141, 834-35). DkTx is unique in that it contains two Inhibitor Cysteine Knots (ICK) motifs connected by a peptide linker. Although these ICK motifs are related to those found in tarantula toxins that target voltage sensors in Kv channels, DkTx does not appear to interact with classical voltage-activated cation channels. We set out to explore the mechanism of DkTx activation of TRPV1, and began by producing the toxin in E. Coli and testing for activity against TRPV1. DkTx was expressed as a fusion with bacterial Ketosteroid Isomerase (KSI), cleaved from KSI using hydroxylamine, and purified using reverse phase HPLC. Reduced DkTx was folded in vitro in a solution containing (GSH/GSSG) and guanidine HCl, and the folding reaction monitored by HPLC. Using this procedure we obtained a predominant species of the toxin that was further purified by reverse phase HPLC. When tested for activity on TRPV1 expressed in Xenopus laevis oocytes, DkTx produced robust and slowly reversible activation of the channel when voltage clamped at À60 mV. At a concentration of 2mM, DkTx produced comparable activation to 2 mM capsaicin, suggesting that the apparent affinity of the recombinant toxin is similar to that reported for the native toxin. We are currently working to solve the structure of DkTx using NMR and further investigating it's mechanism of activation.