Using an External Perfusion System and a Patch Clamp Rig for the Cold Activation of TRPM8

TRPM8 is a member of the transient receptor potential channel (TRP) family. TRPM8 is recognized as a thermosensitive channel, activated by cold temperatures, under ~25 ˚C, and ligands like menthol, Eucalyptol, and icilin1-4.

It is part of the melastatin subfamily of TRP channels5 and demonstrates an outward rectification with moderately high permeability for calcium ions and slight selectivity between monovalent cations. Menthol is secondary alcohol created by the peppermint herb, Mentha piperita. It is commonly utilized in the food and pharmaceutical industries and used as a cooling or soothing compound and odorant.

It activates Ca2+ influx in a subset of sensory neurons from the dorsal root and trigeminal ganglia, where the TRPM8 channel is specifically expressed2-4. It is understood that TRPM8 is responsible for cold allodynia post inflammation5 and it has been described as a potential pharmaceutical target for the treatment of chronic pain and migraine6.

In addition, TRPM8 channels have been seen to play a part in cancer7,8, particularly prostate cancer7 and pancreatic cancer8, and could offer new clinical biomarkers and treatment targets for these forms of cancer.

This article presents data of hTRPM8 gathered on the Port-a-Patch using cooled solution through the External Perfusion System. Capsazepine suppressed cold activated hTRPM8 with an IC50 in strong agreement with the previous research1,9.

Results

Numerous ligands can activate TRPM8 inclusive of the cooling compounds menthol and icilin1-4, in addition to cold temperatures1-4. TRPM8 is a cation channel with voltage-dependence of activation displaying a strong outward rectification at depolarized membrane potentials and rapid, voltage-dependent closure at negative potentials9,10.

TRPM8 -mediated currents are blocked by increased temperatures2,10. Activation of TRPM8 expressed in CHO cells by continuous perfusion of external solution at 10 °C and successive suppression by high temperature, 35 °C, is shown in Figure 1.

(A) Whole-cell current responses from induced CHO cells expressing TRPM8 to a ramp protocol (-100 mV to +100 mV over 200 ms) at 10 °C (dark blue) and 35 °C (light blue). TRPM8 was activated at 10 °C but not at 35 °C. (B) Timecourse of TRPM8 activation with a cold solution (10 °C) and block by warm solution (35 °C).

Figure 1. (A) Whole-cell current responses from induced CHO cells expressing TRPM8 to a ramp protocol (-100 mV to +100 mV over 200 ms) at 10 °C (dark blue) and 35 °C (light blue). TRPM8 was activated at 10 °C but not at 35 °C. (B) Timecourse of TRPM8 activation with a cold solution (10 °C) and block by warm solution (35 °C).

Cooled solution activated TRPM8 and it is possible that this is suppressed by either a higher temperature or by using the blocker capsazepine within the external solution. The application of capsazepine occurred at 10 °C. The time course of the experiment and the corresponding example traces are shown in Figure 2.

(A) Traces from an example cell showing activation by cooled solution (10 °C, dark blue), block by either heated solution (35 °C; light blue), or capsazepine (50 μM; red trace) and washout after capsazepine application (grey). TRPM8 current could be almost completely recovered upon washout of capsazepine. (B) Timecourse of the experiment.

Figure 2. (A) Traces from an example cell showing activation by cooled solution (10 °C, dark blue), block by either heated solution (35 °C; light blue), or capsazepine (50 μM; red trace) and washout after capsazepine application (grey). TRPM8 current could be almost completely recovered upon washout of capsazepine. (B) Timecourse of the experiment.

It was also possible to construct a concentration-response curve for capsazepine using a cooled solution (10 °C) as the activator. Increasing concentrations of capsazepine at 10 °C were applied and Figure 3 shows the concentration-response curve. The IC50 for the capsazepine block of TRPM8 was 12.9 µM (n = 1), in brilliant agreement with the literature1,9.

Concentration response curve for capsazepine using cooled solution to activate TRPM8 reveals an IC50 of 12.9 µM, in good agreement with the literature1,9.

Figure 3. Concentration-response curve for capsazepine using a cooled solution to activate TRPM8 reveals an IC50 of 12.9 μM, in good agreement with the literature1,9.

To summarize, hTRPM8 expressed in CHO cells can be reliably activated utilizing the External Perfusion System of the Port-a-Patch with Temperature Control which allows cooling of the external solution to 10 °C. Cold-activated TRPM8 was also suppressed by capsazepine as anticipated1,9. Therefore, the Port-a-Patch is a helpful tool for recognizing TRPM8 inhibitors as a possible treatment for chronic pain and some cancers.

Methods

Cells

CHO cells stably expressing hTRPM8.

Cell culture

CHO cells stably expressing hTRPM8 were cultured in standard culture conditions for the Port-a-Patch and activated with 1 mg/ml tetracycline for a minimum of 24-36 hours before measurements.

Electrophysiology

Whole-cell patch-clamp recordings were completed in accordance with Nanion’s standard procedure for the Port-a-Patch. Currents were produced by 200 ms voltage ramps from -100 mV to +100 mV, Vhold = -80 mV.

External solution was cooled to 10 °C through the Temperature Control of the External Perfusion System to induce hTRPM8. Capsazepine concentrations were created in external solution and applied to the cell at 10°C through the External Perfusion System of the Port-a-Patch.

Acknowledgments

Produced from materials originally authored by the electrophysiology team at Nanion Technologies GmbH, Munich.

References and Further Reading

  1. Behrendt, H.J., et al., 2004. Br. J. Pharmacol. 141, 737– 745
  2. McKemy, D.D., et al., 2002. Nature 416: 52-58
  3. Peier, A.M., et al., 2002. Cell 108: 705-715
  4. Andersson, D.A., et al., 2004. J. Neurosci. 24(23):5364– 5369
  5. Weyer, A.D. & Leyto, S.G. 2017. Pharmaceuticals. 10: 37
  6. González-Muñiz, R., et al. 2019. Int. J. Mol. Sci. 20: 2618
  7. Hantute-Ghesquier, A. et al. 2018. Pharmaceuticals. 11: 58
  8. Yee, N.S. et al., 2012. Scientifica. 2012: Article 415158
  9. Malkia, A., et al., 2009. Mol. Pain. 6: 62
  10. Nilius, B., et al. 2005. J. Physiol. 567.1: 35–44

About Nanion Technologies

Nanion Technologies is a spin-off from the Center of Nanoscience (CeNS) of the University of Munich (LMU).

Nanion combines bio- and microtechnology in a company serving the life sciences industry by offering products and services which will dramatically increase the speed and efficiency of the drug discovery process in an important segment of the pharmaceutical market.

Nanion bases its business on a proprietary chip technology and will design and develop High Throughput Screening (HTS) systems for ion channel active drugs (ICADs). Ion channels are prime targets for innovative medicines aimed at many important diseases.


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Last updated: Jun 16, 2020 at 3:12 AM

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