Allosteric modulation of vanilloid receptor 1 channel gating by protons
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The recently cloned TRPV1 receptor is responsive to various types of noxious stimuli. The channel is independently activated by capsaicin at sub-micromolar range, by heat above 40 °C, and by protons at pH below 6. Furthermore, simultaneous applications of two or more stimuli lead to cross sensitization of the receptor, with an apparent increase in the sensitivity to any individual stimulus when applied alone. We studied here the mechanism underlying cross-sensitization between capsaicin and pH, the two prototypical stimuli for TRPV1 channel. By analyzing single channel currents recorded from excised-patches expressing single recombinant TRPV1 channels, we examined the effect of pH on burst properties of capsaicin activation at low concentrations and the effect on gating kinetics at high concentrations. Our results suggest that pH has dual effects on both capsaicin binding and channel gating. Lowering pH enhances the apparent binding affinity of capsaicin, promotes the occurrences of long openings and short closures, and stabilizes the open conformation of the channel. We also demonstrated that capsaicin binding and protonation interact allosterically---in a way the effect of one can be offset by the effect of the other. These results provide the basis to further understand the nature of the activation pathways of the channel as well as the general mechanisms underling the cross-sensitizations among other stimuli. A severe acidification leads to direct activation of the TRPV1 receptor in the absence of other stimuli. There is evidence suggesting that these effects may be mechanistically distinct. We showed that replacements of the extracellular pore region between S5 and S6 of TRPV1 with that of TRPV2 eliminated the pH response without diminishing capsaicin activity. A segment of 15 amino acid residues, in the linker between S5 and the pore helix, was not required for the functions of TRPV1. The homology modeling of TRPV1 deleted with 15 residues guaranteed the structural stability of the mutant. We provide here evidence in support of segregation of proton-evoked activation pathway from that involved in capsaicin activation. In search for the molecular basis underlying the direct activation of the channel by low pH, we localized a region (627-634) on the first half of the putative pore helix that is required for the low pH, but not for capsaicin activation. Replacing the region with its counterpart from TRPV1, a proton-insensitive homolog of TRPV2, led to complete abrogation of low pH activity, without significantly altering capsaicin currents. The pore helix region in S5-S6 was mainly responsible for proton gating effect and the residual proton current resulted from E600-mediated potentiation of the heat response. Two amino acid residues, N628 and T633, in the putative pore helix region of the TRPV1 were identified as participants in the proton-gated mechanism.