Multimodal gating is an essential feature of many TRP ion channels, enabling them to respond to complex cellular environments. TRPV1, a pain receptor involved in nociception at the peripheral nerve terminals, can be activated by a range of physical and chemical stimuli (e.g., capsaicin, proton, and heat) and further sensitized by proinflammatory substances. How a single receptor achieves this multiplicity of functionality is poorly understood at the molecular level. Here, we investigated the structural basis of proton activation of TRPV1. Chimeric channels between rTRPV1 and the low pH-insensitive homolog TRPV2 were constructed by systematically exchanging the extracellular domains and were characterized using whole-cell recording in transiently transfected HEK293 cells. Two discrete domains, one involving the pore helix and the other the S3-S4 linker, were found crucial for direct activation of the channel by low pH. Single residue mutations in either domain (T633A/V538L) abrogated the proton-evoked current while preserving the capsaicin and heat responses and their potentiation by mildly acidic pH. Both residues exert a gating effect through hydrophobic interactions. Our results unravel novel information on the structural basis of channel function, and support the existence of discrete domains for multimodal gating of the channel. In view of the resemblance of the pore of TRPV1 to KcsA, our findings also provide evidence on the pore helix as an active component in channel gating in addition to its role in ion permeation.
Capsaicin ion channels are highly expressed in peripheral nervous terminals and involved in pain and thermal sensations. One characteristic of the cloned VR1 receptor is its multimodal responses to various types of noxious stimuli. The channel is independently activated by capsaicin and related vanilloids at submicromolar range, by heat above 40°C, and by protons at pH below 6.5. 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 such cross-sensitization; in particular, between capsaicin and pH, two prototypical stimuli for the channel. By analyzing single-channel currents recorded from excised-patches expressing single recombinant VR1 receptors, 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 indicate 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 at least one of the open conformations of the channel. Our data also demonstrate that capsaicin binding and protonation of the receptor interact allosterically, where the effect of one can be offset by the effect of the other. These results provide important basis to further understand the nature of the activation pathways of the channel evoked by different stimuli as well as the general mechanism underling the cross-sensitization of pain.
HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K+ channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K+ channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd2+ ions freeze the open–closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd2+ and measure the effect on voltage sensor movement. Cd2+ did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd2+-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open–closed equilibrium, corresponding to a coupling energy of ∼1.3–2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K+ channels, but also much weaker.
The vanilloid receptor TRPV1, previously known as VR1, has been implicated in pain sensation under both physiological and pathological conditions. The channel is highly expressed in sensory ganglion neurones and is activated by a range of noxious stimuli including irritant chemicals, acids and heat. In order to understand the structural basis underlying this polymodal activation and the regulation by intracellular signalling pathways, we have investigated the functional roles of the cytoplasmic C-terminal of rat TRPV1. A mutant with the maximal truncation of the distal C-terminal encompassing the last 88 residues was constructed. Of interest, this mutant exhibited a Ca 2+ -dependent functional loss; it was irresponsive to capsaicin in the presence of extracellular Ca 2+ , but fully functional otherwise. Further studies of this construct revealed that extracellular Ca 2+ alone could activate the channel, and that the activation required protein kinase C (PKC) phosphorylation at S502, an event that was up-regulated by external Ca 2+ entry. We compared the truncation mutant with wild-type TRPV1 and demonstrated that it had a significantly increased sensitivity to PKC phosphorylation. These results suggest the distal C-terminal of TRPV1 can inhibit phosphorylation-induced potentiation of the wild-type channel. They also call into question some established functions of the distal C-terminal of TRPV1, including its roles in agonist binding and functional desensitization. We suggest that the functional loss of the truncation mutant, in the presence of extracellular Ca 2+ , was not due to disruption of agonist binding or gating, but rather to desensitization promoted by unstimulated extracellular Ca 2+ entry.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.