Functional Characterization of the Double Knot Toxin (DkTx) as an Activator of the TRPV1 Ion Channel

Abstract

The double knot toxin (DkTx), a peptide isolated from the Chinese bird spider venom, activates the TRPV1 ion channel by binding to the channel with high affinity. As its name suggests, DkTx consists of two cystine-rich lobes (K1 and K2) attached to each other via an oligopeptide linker. These lobes belong to the inhibitory cystine knot (ICK) family of peptide toxins that modulate the activity of voltage-gated ion channels. The mechanism by which ICK peptides activate voltage-gated channels have been well-studied and these peptides have served as powerful pharmacological tools for elucidation of the mechanism of ion channel-mediated voltage sensing. It is therefore tempting to speculate that DkTx has the potential to serve as a powerful tool to help elucidate the hitherto elusive mechanisms of activation of TRPV1 by a wide range of stimuli including chemical ligands and temperature. Here, we outline our initial efforts towards achieving this goal. Specifically, we describe the high-yielding production of DkTx from an E. coli expression system and report voltage clamp experiments that yielded dose response curves for TRPV1 activation by DkTx, K1, and K2, thereby revealing the order of potency of the toxins (DkTx>K2>>K1). Moreover, the dose response curve for K2 suggests a much more cooperative binding of the toxin to the channel (Hill coefficient ∼4) as compared to DkTx (Hill coefficient ∼1). To understand the molecular basis of these results, we are currently studying the activation of TRPV1 by mutants of K1, K2, and DkTx.