The activation mechanism of rat vanilloid receptor 1 by capsaicin involvesthe pore domain and differs from the activation by either acid or heat

The recently cloned rat vanilloid receptor, VR1, can be activated by capsai cin, acid, and heat. To determine the molecular mechanisms facilitating cha nnel opening in response to these stimuli, VR1 and six channels containing charge neutralization point mutations surrounding the putative channel pore domain were expressed and characterized in Xenopus laevis oocytes. Steady- state dose-response relationships, current-voltage relationships, ionic sel ectivities, and single-channel properties were recorded using voltage-clamp techniques. Three of the mutant channels are significantly more sensitive to capsaicin than is wild-type VR1, whereas none differed in their activati on by acidic: pH or temperature. Furthermore, one of the mutants has lost a ll positive cooperativity for capsaicin activation (Hill coefficient congru ent to 1, VR1 congruent to 2), is much more selective for Ca2+, and exhibit s a lower efficacy for acid than for capsaicin activation. Single-channel r ecordings show that capsaicin- and acid-activated channels have the same co nductance, that the three mutants with increased capsaicin sensitivity exhi bit higher open probabilities at submaximal capsaicin concentrations, and t hat the gating properties of capsaicin activation differ from those of acid activation. These data indicate that VR1 undergoes conformational changes upon capsaicin binding that it does not undergo in response to activation b y protons or thermal stimuli. Furthermore, these structural rearrangements include the putative pore domain and reveal the location of an intracellula r domain that contributes to the positive cooperativity seen for capsaicin activation.