The transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific nonselective cation channel that is gated in response to various noxious stimuli: pungent vanilloids, low pH, noxious heat, and depolarizing voltages. By its analogy to K+ channels, the S6 inner helix domain of TRPV1 (Y666-G683) is a prime candidate to form the most constricted region of the permeation pathway and might therefore encompass an as-yet-unmapped gate of the channel. Using alanine-scanning mutagenesis, we identified 16 of 17 residues, that when mutated affected the functionality of the TRPV1 channel with respect to at least one stimulus modality. T670A was the only substitution producing the wild-type channel phenotype, whereas Y666A and N676A were nonfunctional but present at the plasma membrane. The periodicity of the functional effects of mutations within the TRPV1 inner pore region is consistent with an alpha-helical structure in which T670 and A680 might play the roles of two bending "hinges."
- MeSH
- alanin MeSH
- buněčné linie MeSH
- elektrofyziologie MeSH
- financování organizované MeSH
- gating iontového kanálu fyziologie MeSH
- kapsaicin farmakologie MeSH
- kationtové kanály TRPV fyziologie genetika chemie účinky léků MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- membránové potenciály MeSH
- mutace MeSH
- mutageneze cílená MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
We have previously reported that the reducing agent dithiothreitol (DTT) strongly increases thermally induced activity of the transient receptor potential vanilloid receptor-1 (TRPV1) channel. Here, we show that exposure to oxidizing agents also enhances the heat-induced activation of TRPV1. The actions of sulfhydryl modifiers on heat-evoked whole-cell membrane currents were examined in TRPV1-transfected human embryonic kidney 293T cells. The sensitizing effects of the membrane-permeable oxidizing agents diamide (1 mM), chloramine-T (1 mM), and the copper-o-complex (100:400 microM) were not reversed by washout, consistent with the stable nature of covalently modified sulfhydryl groups. In contrast, the membrane-impermeable cysteine-specific oxidant 5,5'-dithio-bis-(2-nitrobenzoic acid) (0.5 mM) was ineffective. The alkylating agent N-ethylmaleimide (1 mM) strongly and irreversibly affected heat-evoked responses in a manner that depended on DTT pretreatment. Extracellular application of the membrane-impermeable reducing agent glutathione (10 mM) mimicked the effects of 10 mM DTT in potentiating the heat-induced and voltage-induced membrane currents. Using site-directed mutagenesis, we identified Cys621 as the residue responsible for the extracellular modulation of TRPV1 by reducing agents. These data suggest that the vanilloid receptor is targeted by redox-active substances that directly modulate channel activity at sites located extracellularly as well as within the cytoplasmic domains. The results obtained demonstrate that an optimal redox state is crucial for the proper functioning of the TRPV1 channel and both its reduced and oxidized states can result in an increase in responsiveness to thermal stimuli.
- MeSH
- buněčné linie MeSH
- diamid farmakologie MeSH
- dithiothreitol farmakologie MeSH
- ethylmaleimid farmakologie MeSH
- financování organizované MeSH
- kapsaicin farmakologie MeSH
- kationtové kanály TRPV fyziologie genetika MeSH
- krysa rodu rattus MeSH
- kyselina dithionitrobenzoová farmakologie MeSH
- lidé MeSH
- membránové potenciály účinky léků MeSH
- metoda terčíkového zámku MeSH
- missense mutace genetika MeSH
- mutace genetika MeSH
- mutantní proteiny fyziologie genetika MeSH
- oxidancia farmakologie MeSH
- peroxid vodíku farmakologie MeSH
- redukční činidla farmakologie MeSH
- sulfhydrylová reagencia farmakologie MeSH
- transfekce MeSH
- vysoká teplota MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- MeSH
- bolest patofyziologie MeSH
- buněčný převod mechanických signálů genetika MeSH
- finanční podpora výzkumu jako téma MeSH
- iontové kanály antagonisté a inhibitory fyziologie chemie MeSH
- kapsaicin MeSH
- molekulární struktura MeSH
- nociceptory klasifikace metabolismus MeSH
- přehledová literatura jako téma MeSH
- senzorická ganglia MeSH
