Elucidating the role of the linker in DkTx mediated TRPV1 activation Mechanism

Abstract

Peptide toxins produced by venomous animals serve as powerful tools for unraveling the mechanisms of modulation of ion channel proteins they target. My thesis work focuses on studying the mechanism of activation of the transient receptor potential vanilloid 1 (TRPV1) ion channel by the double-knot toxin (DkTx), a toxin produced by the Chinese bird spider. Specifically, my work focuses on examining the as yet unexplored role of a seven residues-long “linker” region present in the toxin by generating variants of residues comprising this region of the toxin followed by their electrophysiological characterization as TRPV1 agonists. Three types of toxin variants have been generated—site-directed substitutions of proline residues of the linker, truncations of the linker and substitution of all the residues of the linker to glycine residues. Functional characterization of these variants reveals that whereas proline substituents demonstrate negligible effects on toxin-activation of the channel, truncating the linker results in a profound alteration of both the wash-off rates and the potency of the toxin. Substituting all the linker residues with glycine results in a moderate effect on toxin activity. Taken together, these results demonstrate that the chemistry of the residues comprising the linker plays a minimal role in the toxin’s activity and that the linker primarily serves to provide the separation required for the two knots to reach out and concomitantly dock onto their binding sites on the channel.