Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jun 21;10(25):eadm9228.
doi: 10.1126/sciadv.adm9228. Epub 2024 Jun 21.

Evidence that the cold- and menthol-sensing functions of the human TRPM8 channel evolved separately

Dustin D Luu  1 , Nikhil Ramesh  2 , I Can Kazan  2 , Karan H Shah  1 , Gourab Lahiri  3 , Miyeko D Mana  3 , S Banu Ozkan  2 , Wade D Van Horn  1
Affiliations

Evidence that the cold- and menthol-sensing functions of the human TRPM8 channel evolved separately

Dustin D Luu et al. Sci Adv. .

Abstract

Transient receptor potential melastatin 8 (TRPM8) is a temperature- and menthol-sensitive ion channel that contributes to diverse physiological roles, including cold sensing and pain perception. Clinical trials targeting TRPM8 have faced repeated setbacks predominantly due to the knowledge gap in unraveling the molecular underpinnings governing polymodal activation. A better understanding of the molecular foundations between the TRPM8 activation modes may aid the development of mode-specific, thermal-neutral therapies. Ancestral sequence reconstruction was used to explore the origins of TRPM8 activation modes. By resurrecting key TRPM8 nodes along the human evolutionary trajectory, we gained valuable insights into the trafficking, stability, and function of these ancestral forms. Notably, this approach unveiled the differential emergence of cold and menthol sensitivity over evolutionary time, providing a fresh perspective on complex polymodal behavior. These studies provide a paradigm for understanding polymodal behavior in TRPM8 and other proteins with the potential to enhance our understanding of sensory receptor biology and pave the way for innovative therapeutic interventions.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.. Ancestral TRPM8 reconstruction.
The human evolutionary timeline from the emergence of vertebrates (left) was estimated from TimeTree5. AlphaFold2 models of full-length hTRPM8 (extant, blue). Residue changes from hTRPM8 identified in ASR shown for ancestral primate (AncPrim, purple), ancestral mammalian (AncMam, green), and ancestral vertebrate (AncVert, orange).
Fig. 2.
Fig. 2.. Resurrected ancestral TRPM8 orthologs traffic and function in human cells.
(A) Resurrected ancestral TRPM8 orthologs traffic to the plasma membrane shows similar trafficking. Representative contours in logarithmic scale of flow cytometry data for the AncPrim (purple), AncMam (green), and AncVert (orange) against extant (blue). The number of cells used to make the contours was ungated and normalized to the lowest cell count of 12,607. The average ratio of Alexa Fluor 647 (AF647):enhanced green fluorescent protein (EGFP) are from EGFP+ gated cells with ratios and SD of 0.82 ± 0.10, 0.81 ± 0.9, 0.74 ± 0.11, and 0.76 ± 0.11 for extant, AncPrim, AncMam, and AncVert, respectively. The averages are from six biological replicates conducted on different days, and individual averages of each replicate are jittered. (B) Thermal stability of ancestral TRPM8 orthologs. Representative CETSA Western blots using anti-HA antibody and enhanced chemical luminescence. Combined stability curves show minor differences in stability between the ancestral TRPM8 orthologs with a modest 2°C shift in melting temperature (Tm) when compared to extant (48.0 ± 0.3°C) and AncVert (48.9 ± 0.3°C) with AncPrim (46.1 ± 0.3°C) and AncMam (46.5 ± 0.5°C). Four or five biological sets of CETSA-independent experiments were carried out, with experiments conducted on different days. Errors are in SEM. (C) Patch-clamp electrophysiology of TRPM8 in the absence (top) and presence (bottom) of the cognate TRPM8 agonist menthol (500 μM) indicates that it is a functional ion channel. These trafficking, stability, and functional studies were conducted in HEK-293 cells. Statistical analyses are listed in table S4.
Fig. 3.
Fig. 3.. Thermosensitivity of ancestral TRPM8 orthologs.
Temperature-dependent whole-cell patch-clamp electrophysiology measurements show step-wise decreases in thermosensitivity for ancient TRPM8 orthologs (left). Current density was recorded from temperature ramps from 28° to 8°C at 60 mV and subtracted against the empty pIRES2 vector to remove any endogenous temperature effects (fig. S7A). The data are the averages of 8, 9, 11, and 8 biological replicates for extant (blue), AncPrim (purple), AncMam (green), and AncVert (orange), respectively. Quantification of the cold thermosensitivity (|ΔH|) with SE as a function of evolutionary time indicates that TRPM8 cold sensitivity emerged later in the human evolutionary trajectory (right). Statistical analyses are listed in table S4.
Fig. 4.
Fig. 4.. Ancestral TRPM8 orthologs are menthol receptors with variable menthol affinity.
(A) Menthol response curves with SEM of the ancestral TRPM8 orthologs from calcium mobilization assay with addition of menthol. (B) The menthol half-maximal effective concentration (EC50) (potency) was calculated from Fluoforte-based Ca2+ flux data of nine biological replicates with the values plotted with SE. All EC50 values had a P value of <0.0001 with each other using an unpaired t test. (C) The relative menthol efficacy (E) with SEM of ancestral TRPM8 orthologs was estimated from current-density fold increase from patch-clamp measurements in the absence and presence of 500 μM menthol. (D) E varies linearly (R2 = 0.98) with EC50 indicative of variable affinity (Kd), where AncMam (green) has the highest relative affinity, potency, and efficacy for menthol. (E) The V1/2 with SE of the voltage-menthol coupling obtained from the whole-cell patch-clamp tail currents. (F) V1/2 shows a linear relationship (R2 = 0.99) with the emergence of extant, AncPrim, and AncMam TRPM8. Whole-cell patch-clamp electrophysiology data are from 8 to 11 biological replicates collected over multiple days and transfections. Statistical analyses are listed in table S4.
Fig. 5.
Fig. 5.. Temperature-dependent differences in calculated flexibility.
(A) The difference between normalized DFI (ΔDFI) calculated at 27° and 10°C for extant, AncPrim, AncMam, and AncVert TRPM8 highlights regions with predicted temperature-dependent dynamics. The data represent the means ± SEM of the chain-wise ΔDFI. (B) The change in flexibility colored on the structure for all extant and ancestral TRPM8 orthologs. Regions colored red show increased flexibility on heating, and regions colored blue show increased flexibility on cooling.
Fig. 6.
Fig. 6.. Relationship between TRPM8 thermosensitivity and menthol sensitivity with evolutionary time.
An inverse relationship between cold (blue) and menthol sensitivity (gray) along the hTRPM8 evolutionary trajectory. ASR enables the dissection of TRPM8 polymodal activation. Data points are connected via spline interpolation.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. McKemy D. D., Neuhausser W. M., Julius D., Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52–58 (2002). - PubMed
    1. Peier A. M., Moqrich A., Hergarden A. C., Reeve A. J., Andersson D. A., Story G. M., Earley T. J., Dragoni I., McIntyre P., Bevan S., Patapoutian A., A TRP channel that senses cold stimuli and menthol. Cell 108, 705–715 (2002). - PubMed
    1. Hensel H., Zotterman Y., The effect of menthol on the thermoreceptors. Acta Physiol. Scand. 24, 27–34 (1951). - PubMed
    1. Winchester W. J., Gore K., Glatt S., Petit W., Gardiner J. C., Conlon K., Postlethwaite M., Saintot P. P., Roberts S., Gosset J. R., Matsuura T., Andrews M. D., Glossop P. A., Palmer M. J., Clear N., Collins S., Beaumont K., Reynolds D. S., Inhibition of TRPM8 channels reduces pain in the cold pressor test in humans. J Pharmacol Exp Ther. 351, 259–269 (2014). - PubMed
    1. Horne D. B., Biswas K., Brown J., Bartberger M. D., Clarine J., Davis C. D., Gore V. K., Harried S., Horner M., Kaller M. R., Lehto S. G., Liu Q., Ma V. V., Monenschein H., Nguyen T. T., Yuan C. C., Youngblood B. D., Zhang M., Zhong W., Allen J. R., Chen J. J., Gavva N. R., Discovery of TRPM8 Antagonist (S)-6-(((3-fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)carbamoyl)nicotinic acid (AMG 333), a clinical candidate for the treatment of migraine. J. Med. Chem. 61, 8186–8201 (2018). - PubMed