The Stoichiometry and Activation Mechanism of TRPV1 by Vanilloids

Vanilloids are pain evoking molecules that serve as ligands of the ‘heat and capsaicin receptor’ TRPV1. It is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat (>42°C) and various noxious chemicals. TRPV1 channel is formed by the symmetrical arrangement of four identical subunits around a central ion-conducting pore. Binding of vanilloids evokes channel opening and subsequent neuronal activation, leading to pain sensation. Despite its pivotal physiological role, the molecular basis of TRPV1 activation by vanilloids is not fully understood. Here, we combine the use of site-directed mutagenesis, concatemeric constructs harboring mutated binding sites with patch-clamp recordings and calcium imaging in order to determine the stoichiometry and activation mechanism of TRPV1 by the exo-vanilloid capsaicin, the ‘hot’ component of chili peppers. We show that a single capsaicin-bound subunit is sufficient to achieve a maximal open-channel lifetime, while the high sensitivity for this agonist requires all four subunits. Moreover, we defined the role of residues within TRPV1 vanilloid binding site in the channel activation mechanism, by acting to stabilize ligand-receptor interaction.