Lipid-Mediated Interaction of Double-Knot Toxin with TRPV1 Channels

Abstract

The Transient Receptor Potential V1 (TRPV1) channel is a cation channel that opens in response to diverse chemical and physical stimuli. These stimuli include capsaicin, temperature, pH and bioactive lipids. Recently, a novel peptide named Double cystine Knot Toxin (DkTx) was purified from tarantula venom and shown to activate the channel from the extracellar site of the membrane and proposed to bind the pore region (Bohlen CJ., et al, 2010). In order to characterize DkTx-TRPV1 interactions, we expressed and purified recombinant His-tagged DkTx in E. coli and 1D4-tagged TRPV1 in Saccharomyces cerevisiae. Expressed TRPV1 binds the specific TRPV1 agonist, 3H- resiniferatoxin (RTx), with high affinity (Kd∼500 pM) and in the presence of DkTx the affinity of RTx increases by ∼4-fold, indicating that both RTx and DkTx interact with the TRPV1 channel in membranes, but at distinct sites that interact allosterically. Pull-down experiments with full-length and truncated TRPV1 (containing the pore-forming S5-S6 segments and the C-terminus suggest that DkTx interacts with both detergent purified channel constructs, supporting an interaction of the toxin with the external pore region. DkTx and TRPV1 constructs do not, however, co-elute as a complex in size-exclusion or affinity chromatography, suggesting that in detergent solution the interactions are weak. To investigate the strength of TRPV1 interactions with DkTx in detergent micelles we utilized surface plasmon resonance with TRPV1 deposited on a gold chip using 1D4 tag. DkTx interacts with TRPV1 in detergent micelles, but with much lower apparent affinity than observed in membranes. The isolated K2 lobe of DkTx exhibited higher affinity than K1, in agreement with the previous electrophysiology studies. These results support the notion that DkTx interacts with the external pore of TRPV1 and suggest that the membrane lipids play a critical role in toxin-channel interaction.