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

Abstract

The recently cloned rat vanilloid receptor, VR1, can be activated by capsaicin, acid, and heat. To determine the molecular mechanisms facilitating channel 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 doseresponse relationships, current-voltage relationships, ionic selectivities, 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 activation by acidic pH or temperature. Furthermore, one of the mutants has lost all positive cooperativity for capsaicin activation (Hill coefficient Х 1, VR1 Х 2), is much more selective for Ca 2؉ , and exhibits a lower efficacy for acid than for capsaicin activation. Single-channel recordings show that capsaicin-and acid-activated channels have the same conductance, that the three mutants with increased capsaicin sensitivity exhibit higher open probabilities at submaximal capsaicin concentrations, and that 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 by protons or thermal stimuli. Furthermore, these structural rearrangements include the putative pore domain and reveal the location of an intracellular domain that contributes to the positive cooperativity seen for capsaicin activation.C apsaicin, the primary pungent compound in ''hot'' chili peppers, produces pain and inflammation when placed on skin or mucus membranes. These responses are a consequence of capsaicin activating nonselective cation channel receptors within C and A␦ nociceptors and inducing the release of peptides and other transmitters from their peripheral and central terminals (1). Although a number of reports have demonstrated the existence of capsaicin receptors in sensory neurons (2, 3), the cloning of rat vanilloid receptor 1 (VR1) (4) was a breakthrough in understanding their properties and physiological functions (3, 5). VR1 subunits have now been identified in peripheral and central terminals of neurons in sensory ganglia (1,3,4,6,7). Electrophysiological studies of expressed VR1 receptors show that they are activated independently by capsaicin (EC 50 ϭ 0.7 M), acid (pH 50 ϭ 5.4), and elevated temperatures Ն 43°C (3, 6). These conditions are similar to those found in some physiological states (2,3,8). Further findings were that two or more of these activation pathways can act in concert to increase the sensitivity of VR1 receptors to any one stimulus (6,8). To determine protein domains important for the activation of VR1 by capsaicin, protons, and heat, we constructed a number of VR1 point mutants and measured their responsiveness to these three activators. We found that mutations near the pore of VR1 channels ca...

Uncoupling Proton Activation of Vanilloid Receptor TRPV1

“…The involvement of V538 also underscores the importance of the peripheral domains of the channel for proton activation. This extends the previous findings on the acidic residues in the pore region (Jordt et al, 2000;Welch et al, 2000), and suggests a global conformational change evoked by protonation. The latter is consistent with the pharmacological data, which shows that capsazepine, a competitive inhibitor of capsaicin, inhibits both capsaicin-and proton-evoked currents in rat TRPV1.…”

“…Another acidic residue, E648, located in the linker between the selectivity filter and the sixth transmembrane domain, was proposed to mediate the direct response to low pH (Jordt et al, 2000). The residue as well as two other pore acidic residues, E636 and D646, were also suggested to specifically contribute to capsaicin response without affecting proton or thermal sensitivity (Welch et al, 2000). Similar findings were reported for residues on the pore-aligning segment where mutation tended to affect the capsaicin activity more profoundly (Kuzhikandathil et al, 2001).…”

“…Previous studies on the proton sensitivity of TRPV1 have focused on the extracellular acidic residues (Jordt et al, 2000;Welch et al, 2000). To obtain a more complete understanding of the process, we took a chimera approach and investigated the extracellular domains systematically.…”

Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes

“…To better clarify the off-response of TRPV3, we conducted comparative measurements for the off-response of TRPV1 induced by removal of the extracellular acidic solution from outside-out patches (45,46). A small and rapid off-response was observed in TRPV1 after acid exposure (pH 4.5).…”

TRPV1 Recapitulates Native Capsaicin Receptor in Sensory Neurons in Association with Fas-Associated Factor 1

“…Because acid acts on the extracellular surface of TRPV1 (Jordt et al, 2000;Welch et al, 2000), acidic solutions at different degrees of acidity (pH 4.75-7.0) were applied to outside-out membrane patches. This time, CHO cells were chosen for the transfection of TRPV1 or TRPV1 and FAF1 because of the difficulty in forming outside-out patches from HEK 293T cells.…”