Using Different Stimuli on a Patch Clamp Rig for the Activation and Inhibition of TRPV4

The transient receptor potential channel (TRP) family has a member called TRPV4. Transient receptor potential vanilloid type 4, also known as TRPV4, shares around 40% identity with TRPV1 and TRPV21 and is a Ca2+-permeable non-selective cation channel1-3 expressed in a large range of tissues.

This includes central and peripheral nervous system neurons, and in non-neuronal tissue such as human T cells, epithelial cells of the cornea and retina, endothelial cells of the eye, liver, heart, kidney, synoviocytes, the epithelial lining of lung airways and trachea, pancreatic stellate cells, and several more2.

TRPV4 is triggered by many stimuli inclusive of endogenous ligands like lipid arachidonic acid and its metabolites4, synthetic ligands like GSK1016790A5, warm temperatures higher than 27- 35 °C6,7, hypotonic extracellular solution (cell swelling)1,8, and mechanical stress2. Due to its extensive distribution in several organs, suggestions have been made that TRPV4 plays a large part in several physiological processes2,9.

These processes are inclusive of osmoregulation, thermoregulation, Ca2+ homeostasis in vascular endothelium, sustaining vascular tone and endothelial cell function, in addition to playing a part in the cardiac, urinary, respiratory, skeletal, and digestive systems2,3,9.

In addition, TRPV4 is suggested to play a part in the central nervous system through neuronal excitability and nociception2,8. TRPV4 has been connected with several diseases inclusive of neuropathic and inflammatory pain, respiratory and cardiovascular diseases, and cancer2,3,9.

This article presents data of hTRPV4 recorded on the Port-a-Patch using a heated solution through the External Perfusion System. Heat-activated hTRPV4 was suppressed by ruthenium red. The TRPV4 specific agonist, GSK1016790A, also triggered TRPV4 and the response was suppressed by ruthenium red.

Results

TRPV4 can be triggered by several stimuli, inclusive of warm temperatures higher than 27 °C6,7. Researchers in this study activated hTRPV4 expressed in CHO cells through a heated solution at 45 °C. Some basal activity of TRPV4 occurred at room temperature but the current amplitude rose from control current of 2.7 ± 0.6 nM (n = 6; p<0.01, Student’s t-test) to 5.7 ± 0.8 nA (n = 6).

The activation of TRPV4 via warm solution from an example cell and the timecourse of the experiment is shown in Figure 1.

(A) Whole-cell current responses from induced CHO cells expressing TRPV4 to a ramp protocol (-100 mV to +100 mV over 200 ms) at RT (21 °C) (black) and 45 °C (light blue). TRPV4 was activated at 45 °C. Some basal activity was seen in some cells at the start of the experiment. (B) Timecourse of TRPV4 activation with a warm solution (45 °C).

Figure 1. (A) Whole-cell current responses from induced CHO cells expressing TRPV4 to a ramp protocol (-100 mV to +100 mV over 200 ms) at RT (21 °C) (black) and 45 °C (light blue). TRPV4 was activated at 45 °C. Some basal activity was seen in some cells at the start of the experiment. (B) Timecourse of TRPV4 activation with a warm solution (45 °C).

TRPV4 was triggered by a warm solution and suppressed by ruthenium red applied at 45°C. The timecourse of the experiment and the corresponding example traces are shown in Figure 2.

(A) Traces from an example cell showing basal TRPV4 current (black), activation by warm solution (45 °C, light blue), and block by 50 μM ruthenium red (RR; red). (B) Timecourse of the experiment.

Figure 2. (A) Traces from an example cell showing basal TRPV4 current (black), activation by warm solution (45 °C, light blue), and block by 50 μM ruthenium red (RR; red). (B) Timecourse of the experiment.

TRPV4 was also induced by the specific activator, GSK1016790A. Figure 3 demonstrates the timecourse of the experiment where TRPV4 is induced by GSK1016790A and then suppressed by ruthenium red.

Activation of TRPV4 by GSK1016790A and subsequent block by ruthenium red. Shown is the timecourse of an example cell with corresponding traces.

Figure 3. Activation of TRPV4 by GSK1016790A and subsequent block by ruthenium red. Shown is the timecourse of an example cell with corresponding traces.

To summarize, hTRPV4 expressed in CHO cells can be reliably activated utilizing the External Perfusion System of the Port-a-Patch with Temperature Control which allows heating of the external solution up to 50 °C.

TRPV4 was also activated by GSK1016790A. The current was suppressed by ruthenium red irrespective of an activation stimulus, either heat or GSK1016790A. Therefore, the Port-a-Patch is a helpful tool for finding TRPV4 inhibitors as possible treatments for various conditions including neuropathic and inflammatory pain, respiratory and cardiovascular disease, and some cancers.

Methods

Cells

CHO cells stably expressing hTRPV4.

Cell culture

CHO cells stably expressing hTRPV4 were cultured using standard culture conditions for the Port-a-Patch and activated using 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 Porta-Patch. Currents were produced by 200 ms voltage ramps from -100 mV to +100 mV, Vhold = -80 mV.

External solution was heated to 45 °C with the Temperature Control of the External Perfusion System to induce hTRPV4. Alternatively, hTRPV4 was induced by 60 nM GSK1016790A through the external solution. Ruthenium red was created in external solution and applied at 45 °C or co-applied with GSK1016790A at room temperature.

Acknowledgments

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

References and Further Reading

  1. Clapham, D.E., et al., 2005. Pharmacol. Rev. 57:427-450
  2. White, J.P.M., et al., 2016 Physiol. Rev. 96: 911-973
  3. Darby, W.G., et al., 2016 Int. J. Biochem. & Cell Biol. 78: 217–228
  4. Watanabe, H., et al., 2003. Nature. 424: 434-438
  5. Thorneloe, K.S., et al., 2008. JPET 326: 432-442
  6. Tominaga, M. & Caterina, M.J. 2004. Inc. J. Neurobiol. 61: 3-12
  7. Güler, A.D., et al., 2002. J.Neurosci. 22(15): 6408-6414
  8. Liedtke, W., et al., 2000. Cell. 103(3): 525-535
  9. Everaerts, W., et al., 2010. Prog. in Biophys. Mol. Biol. 103: 2-17

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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