Dataset Information

The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module.

ABSTRACT: The TRPV1 cation nonselective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low extracellular pH, high temperature, or naturally occurring pungent molecules such as allicin, capsaicin, or resiniferatoxin. Its noxious thermal sensitivity makes it an important participant as a thermal sensor in mammals. However, details of the mechanism of channel activation by increases in temperature remain unclear. Here, we used a combination of approaches to try to understand the role of the ankyrin repeat domain (ARD) in channel behavior. First, a computational modeling approach by coarse-grained molecular dynamics simulation of the whole TRPV1 embedded in a phosphatidylcholine and phosphatidylethanolamine membrane provides insight into the dynamics of this channel domain. Global analysis of the structural ensemble shows that the ARD is a region that sustains high fluctuations during dynamics at different temperatures. We then performed biochemical and thermal stability studies of the purified ARD by the means of circular dichroism and tryptophan fluorescence and demonstrate that this region undergoes structural changes at similar temperatures that lead to TRPV1 activation. Our data suggest that the ARD is a dynamic module and that it may participate in controlling the temperature sensitivity of TRPV1.

SUBMITTER: Ladron-de-Guevara E

PROVIDER: S-EPMC7036727 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module.

Ladrón-de-Guevara Ernesto E Dominguez Laura L Rangel-Yescas Gisela E GE Fernández-Velasco Daniel A DA Torres-Larios Alfredo A Rosenbaum Tamara T Islas Leon D LD

Biophysical journal 20191111 4

The TRPV1 cation nonselective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low extracellular pH, high temperature, or naturally occurring pungent molecules such as allicin, capsaicin, or resiniferatoxin. Its noxious thermal sensitivity makes it an important participant as a thermal sensor in mammals. However, details of the mechanism of channel activation by increases in temperature remain unclear. Here, we used a combi ...[more]

PMID: 31757360

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Project description:Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound IκBα molecules (0.67) than for the free IκBα molecules (0.74), apparently indicating that binding of NFκB(RelA/p50) stretches the ARD of IκBα. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NFκB(RelA/p50) and IκBα encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on IκBα as well as IκBα bound to dimerization-domain-only constructs or full-length NFκB(RelA/p50). Although we expected the stretched IκBα to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NFκB NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the IκBα ARD induced by interactions of the IκBα proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

Dataset Information

The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module.

Publications

The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module.

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