Application NoteChannel:
Cells:
Tools:hTRPV4
CHO
Port-a-Patch

Activation and inhibition of TRPV4 using
different stimuli on the Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

TRPV4 is a member of the transient receptor potential

TRPV4 can be activated by a number of stimuli, includ-channel (TRP) family. Transient receptor potential vanilloid

ing warm temperature >27°C6,7. We activated hTRPV4

type 4 (TRPV4) shares approximately 40% identity withexpressed in CHO cells using heated solution at 45°C.

TRPV1 and TRPV2 and is a Ca -permeable non-selective

There was some basal activity of TRPV4 at room tempera-cation channel1-3 expressed in a wide range of tissues

ture but the current amplitude increased to 5.7 ± 0.8 nA

including neurons of the central and peripheral nervous(n = 6) from control current of 2.7 ± 0.6 nM (n = 6; p<0.01,

systems, and in non-neuronal tissue including human T

Student’s t test). Figure 1 shows the activation of TRPV4 bycells, corneal and retinal epithelial cells, endothelial cellswarm solution from an example cell and the timecourseof the eye, liver, heart, kidney, synoviocytes, epithelialof the experiment.
ART

12+lining of trachea and lung airways, stellate cells of the45°C

pancreas, and many more2. TRPV4 is activated by anumber of stimuli including endogenous ligands suchas lipid arachidonic acid and its metabolites4, synthetic
ligands such as GSK1016790A5, warm temperature >2735°C6,7, hypotonic extracellular solution (cell swelling)1,8,and mechanical stress2. Given its widespread distribution
in many organs, this suggests that TRPV4 plays a major
osmoregulation, thermoregulation, Ca2+

homeostatus in

vascular endothelium, maintaining vascular tone andendothelial cell function, as well as playing roles in thecardiac, respiratory, urinary, skeletal, and digestive systems2,3,9. Additionally, TRPV4 is thought to play a role inneuronal excitability in the central nervous system andnociception2,8. TRPV4 has been implicated in a number of
diseases including neuropathic and inflammatory pain,respiratory and cardiovascular diseases, and cancer

2,3,9.

Here we present data of hTRPV4 collected on thePort-a-Patch using heated solution via the External
Perfusion System. Heat activated hTRPV4 was blocked byruthenium red. The TRPV4 specific agonist, GSK1016790A
also activated TRPV4 and the response was blocked byruthenium red.
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B

0 nA

45°C
10Amplitude (nA)role in many physiological processes2,9. These include

1 nA
20 ms

86
4RT (21°C)

RT (21°C)

20050150200100Time(s)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 Timecourseof TRPV4 activation with warm solution (45°C).

Application Note
TRPV4 was activated by warm solution and blocked byruthenium red applied at 45°C. Figure 2 shows the timecourse of the exeriment and corresponding example

3.5

3.0
Control
60 nM GSK1016790A
50 µM RR

2.52 nA
20 ms0 nA
B

45°C

850 µM RR

6Amplitude (nA)

50 µM RR

4.0Control (RT)45°C
50 µM RR (45°C)

Amplitude (nA)traces.
Aand then blocked by ruthenium red.
60 nM GSK1016790A
4.5

2.00 nA

1.5
1.01 nA
20 ms

50

100

150

200
250Time (s)

300

350

4Figure 3: Activation of TRPV4 by GSK1016790A and subsequent blockby ruthenium red. Shown is the timecourse of an example cell withcorresponding traces.

2In conclusion, hTRPV4 expressed in CHO cells can bereliably activated using the External Perfusion System0-2of the Port-a-Patch with Temperature Control which0206040Time (s)enables heating the external solution up to 50°C. TRPV480100Figure 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 activated by the specific activator,GSK1016790A. Figure 3 shows the timecourse of theexperiment where TRPV4 is activated by GSK1016790A

References

was also activated by GSK1016790A. The current was
blocked by ruthenium red regardless of activation stimulus (heat or GSK1016790A). The Port-a-Patch is, therefore, a useful tool for identifying TRPV4 inhibitors as potential therapeutics for a variety of conditions including
neupathic and inflammatory pain, respiratory andcardiovascular disease and and some cancers.
Cell culture
CHO cells stably expressing hTRPV4 were cultured using

1. Clapham, D.E., et al., 2005. Pharmacol. Rev. 57:427-450

standard culture conditions for the Port-a-Patch and

2. White, J.P.M., et al., 2016 Physiol. Rev. 96: 911-973

induced using 1 mg/ml tetracycline for at least 24-36

3. Darby, W.G., et al., 2016 Int. J. Biochem. & Cell Biol. 78:

hours prior to measurements.

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

Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for the Porta-Patch. Currents were elicited by 200 ms voltage ramps
from -100 mV to +100 mV, Vhold = -80 mV. External
solution was warmed 45°C using the Temperature Controlof the External Perfusion System to activate hTRPV4.
Alternatively,hTRPV4

wasactivated

by60

nM

Methods

GSK1016790A via the external solution. Ruthenium redCells

co-applied with GSK1016790A at room temperature.

was made up in external solution and applied at 45°C orCHO cells stably expressing hTRPV4.
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