Pore region of TRPV3 ion channel is specifically required for heat activation

Abstract

Ion channels can be activated (gated) by a variety of stimuli, including chemicals, voltage, mechanical force or temperature. Although molecular mechanisms of ion channel gating by chemical and voltage stimuli are understood in principal, the mechanisms of temperature activation remain unknown. The transient receptor potential channel TRPV3 is a nonselective cation channel that is activated by warm temperatures and sensory chemicals such as camphor. Here we screened 14,000 random mutant clones of mouse TRPV3 and identified five single point mutations that specifically abolish heat activation but do not perturb chemical activation or voltage modulation. Notably, all five mutations are located in the putative sixth transmembrane helix and the adjacent extracellular loop in the pore region of mouse TRPV3. Although distinct in sequence, we found that the corresponding loop of frog TRPV3 is also specifically required for heat activation. These findings demonstrate that the temperature sensitivity of TRPV3 is separable from all other known activation mechanisms and implicate a specific region in temperature sensing.

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Figure 1: TRPV3 mutant library screen.
Figure 2: Concentration responses and heat activation curves for single-point mutants.
Figure 3: Electrophysiology of heat sensitivity of single-point mutants Ile644Ser, Asn647Tyr, Tyr661Cys and wild-type TRPV3.
Figure 4: Basic channel properties of Ile644Ser, Asn647Tyr, Tyr661Cys and wild-type TRPV3.
Figure 5: Location of the heat-sensitive domain.
Figure 6: Xenopus TRPV3 and TRPV chimeric channels.

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Acknowledgements

We thank A.J. Wilson and J. Mainquist for manufacturing the temperature device, A. Marelli and T. Orth for preparing miniprep DNA and M. Caterina for providing rat TRPV1 plasmid DNA. We thank T. Bartfai, E. Lattman, I. MacRae and A. Dubin for helpful discussion. This research was supported by grants from the US National Institutes of Health and by the Novartis Research Foundation.

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J.G. and H.H. contributed equally to this work. J.G. and H.H. designed the study, collected and analyzed data and wrote the manuscript. M.B. participated in designing the temperature device. B.B. conducted molecular modeling. M.S. carried out the biochemical experiments. M.P. participated in producing the random mutant library. A.P. designed the study and wrote the manuscript. All authors discussed results and commented on the manuscript.

Corresponding author

Correspondence to Ardem Patapoutian.

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Supplementary Figures 1–3 and Supplementary Tables 1–4

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Grandl, J., Hu, H., Bandell, M. et al. Pore region of TRPV3 ion channel is specifically required for heat activation. Nat Neurosci 11, 1007–1013 (2008). https://doi.org/10.1038/nn.2169

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