Exploring the Architecture of the Outer Pore of the TRPV1 Channel with Double-Knot Toxin

Abstract

The TRPV1 channel is a non-selective homotetrameric cation channel that acts in nociceptors as a sensor for external noxious chemicals and physical stimuli. It is also modulated by many cell-signaling molecules whose concentrations increase during inflammation and tissue damage. As a consequence, its function has been associated with inflammatory hyperalgesia and neuropatic pain. At present, little structural or mechanistic information is available for this protein. However, the pore domain is thought to play a fundamental role in channel function, since modulating signals are expected to converge on the pore to gate ion permeation. There is also growing evidence to suggest that the pore is the site of action of protons, possibly temperature, and the double ICK knot tarantula toxin (DkTx). DkTx, or its isolated single knots, have been proposed to bind to the external pore to promote activation. Here, we set out to study DkTx-TRPV1 interactions to gain structural information on the pore domain. By using a concatenated TRPV1 channel tetramer, in which toxin-activation was disrupted in specific subunits, we found that the binding of a single toxin knot is sufficient to promote channel activation. We also found that DkTx activates channels in which toxin-activation has been disrupted in either adjacent or opposite subunits, indicating that the knots of DkTx can arrange differently to activate the TRPV1 channel. Finally, we found that a hexa-histidine tag attached to the N-terminus of the toxin acts a voltage- and pH-dependent blocker. We are currently using variants of DkTx, in combination with concatenated TRPV1 channels, to delineate the toxin binding site on the external surface of the channel relative to the central pore and to probe agonist-dependent conformational changes in the pore.