Product Data Sheet hTRPV4-CHO Catalog Number

Product Data Sheet 2010
Page 1 of 10
Stably Transfected Cell Line - Product Data Sheet
hTRPV4-CHO
Catalog Number CT6194
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hTRPV4-CHO
Catalog # CT6194
Product Data Sheet 2010
Page 2 of 10
hTRPV4-CHO
Catalog # CT6194
Table of Contents
1
Cell Line Description.............................................................................................................. 3
1.1
Background ..................................................................................................................... 3
1.2
Pore-forming subunit identifier: hTRPV4 ...................................................................... 3
1.3
Sequence Information ..................................................................................................... 3
1.4
Expression System .......................................................................................................... 3
1.5
Product Format................................................................................................................ 3
1.6
Mycoplasma Status: Negative......................................................................................... 3
1.7
Cell Line Stability ........................................................................................................... 3
2
Validated Test Platforms......................................................................................................... 3
2.1
Manual Patch Clamp Representative Data ..................................................................... 4
2.2
FLIPR Tetra® Representative Data................................................................................. 6
2.2.1
TRPV4 activation................................................................................................ 6
2.2.2
Effects of TRPV4 Antagonists............................................................................ 9
3
References............................................................................................................................. 10
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
Product Data Sheet 2010
Page 3 of 10
1
hTRPV4-CHO
Catalog # CT6194
Cell Line Description
1.1 Background
TRPV4 (transient receptor potential channel, subtype V4) is selective (PCa/PNa = 6),
calcium-permeable cation channel, expressed in peripheral nerve. TRPV4 channels are
activated by the endogenous neurochemical anandamide (endovanilloid) and increased cell
volume in hypotonic media. TRPV4 channels have potential therapeutic significance in
treatment of pain and asthma.
1.2 Pore-forming subunit identifier: hTRPV4
Class: Transient Receptor Potential V (Vanilloid)
Species: Human
Gene Name: TRPV4
1.3 Sequence Information
The cDNA sequence of the hTRPV4 gene used to create this cell line was confirmed prior
to transfection. The amino acid sequence encoded by the transfected cDNAs was identical
to the translated sequence for GenBank accession number NM_021625.3.
1.4 Expression System
CHO (Chinese hamster ovary) cells, tetracycline-inducible.
1.5 Product Format
Cryopreserved cells, 1 x106 cells/vial.
1.6 Mycoplasma Status: Negative
The absence of mycoplasma species in this cell line was confirmed with the MycoAlert Kit
(Lonza Rockland, Inc.).
1.7 Cell Line Stability
Channel expression has been found to be stable for at least 31 passages.
2
Validated Test Platforms
Electrophysiological and pharmacological verification of the functional properties of the cloned
channels were assessed using the following test platforms:
Manual Patch Clamp
FLIPR® (MDS-AT)
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
Product Data Sheet 2010
Page 4 of 10
hTRPV4-CHO
Catalog # CT6194
2.1 Manual Patch Clamp Representative Data
vehicle
A
1 µM 4α-PDD
1 µM 4α-PDD +1 mM La3+
0
Current (pA)
-2000
-4000
-6000
-8000
0
500
1000
Time (s)
1500
2000
B
0
Current (pA)
-1000
vehicle
1 µM 4α-PDD
vehicle
-2000
-3000
-4000
0
500
1000
Time (s)
1500
2000
Figure 1. Time Course of 4-αPDD-Activation of hTRPV4 in Manual Patch
Clamp.
A: Inward currents were elicited by application of 1 µM 4α-PDD (4α-phorbol 12,13didecanoate) in CHO cells transfected with hTRPV4 cDNA at a test potential of
-70 mV. Current amplitude was completely inhibited after 1 mM La3+.
B: Untransfected cells were insensitive to 4α-PDD.
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
Product Data Sheet 2010
Page 5 of 10
hTRPV4-CHO
Catalog # CT6194
100%
Percent Inhibition
80%
60%
40%
20%
0%
0.01
0.1
1.0
10
100
1000
Concentration (µM)
Figure 2. Concentration-Response Relationship for La3+ Inhibition of TRPV4
Current in Manual Patch Clamp.
hTRPV4 channels activated by 1 µM 4α-PDD. Percent block of hTRPV4 current
after application of 0.01, 0.1, 1, 10, 100 and 1000 µM La3+ Mean ± SEM, n = 3
cells/concentration. IC50 = 1.1 µM.
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
Product Data Sheet 2010
Page 6 of 10
hTRPV4-CHO
Catalog # CT6194
2.2 FLIPR Tetra® Representative Data
2.2.1 TRPV4 activation
4α-PDD
(µM)
0
0.01
0.03
0.1
0.3
1
3
10
Untransfected CHO
hTRPV4-CHO
Figure 3. 4α-PDD Activation of hTRPV4-CHO Cells
TRPV14 cells (columns 7-12) showed 4α-PDD-induced increases in [Ca2+]i.
Untransfected CHO cells (columns 1 - 6) were unresponsive to the agonist.
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
Product Data Sheet 2010
Page 7 of 10
hTRPV4-CHO
Catalog # CT6194
4α-PDD
(µM)
0
0.01
0.03
0.1
0.3
1
3
10
Figure 4. 4α-PDD Concentration-Dependence
Application of increasing concentrations of the agonist, 4α-PDD elicited
concentration-dependent increase in [Ca2+]i.signals.
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
Relative Fluorescence Units
Product Data Sheet 2010
Page 8 of 10
hTRPV4-CHO
Catalog # CT6194
4000
3000
2000
1000
0
0.01
0.1
1.0
10
100
Concentration (µM)
Figure 5. 4α-PDD Concentration-Response Relationship.
Mean ± SEM, n = 6 - 12 replicates/concentration. EC50 = 0.64 µM.
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
1000
Product Data Sheet 2010
Page 9 of 10
hTRPV4-CHO
Catalog # CT6194
2.2.2 Effects of TRPV4 Antagonists
La3+
(mM)
RR (µM)
0.0
0.0
0.003
0.01
0.01
0.03
0.03
0.1
0.1
0.3
0.3
1.0
1.0
3.0
3.0
10.0
La3+
Ruthenium Red
Figure 6. Antagonist-Induced Inhibition of TRPV4 in FLIPR®
La3+ (columns 1 - 6) or ruthenium red (columns 7 - 12) at increasing concentrations
inhibited [Ca2+]i signals in 4α-PDD (1 µM)-activated TRPV4 channels in a
concentration-dependent manner.
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.
Product Data Sheet 2010
Page 10 of 10
hTRPV4-CHO
Catalog # CT6194
A
Percent Inhibition
90%
70%
50%
30%
10%
0.001
0.01
0.1
1.0
10
La3+ Concentration (mM)
B
Percent Inhibition
90%
70%
50%
30%
10%
0.001
0.01
0.1
1.0
10
Ruthenium Red Concentration (µM)
Figure 7. La3+ and Ruthenium Red Concentration-Response Relationships.
Inhibition of [Ca2+]i signals in 4α-PDD (1 µM)-activated TRPV4 channels. Mean ±
SEM, n = 18 replicates/concentration. La3+ IC50 = 0.42 mM; ruthenium red IC50 =
0.09 µM.
3
References
Clapham DE, et al. 2005. International Union of Pharmacology. XLIX. Nomenclature and
structure-function relationships of transient receptor potential channels. Pharmacol Rev 57: 427 450.
Watanabe H, et al. 2002. Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol
derivatives. J Biol Chem 277:13569-13577.
© 2010 ChanTest Corporation, Cleveland, OH 44128. All rights reserved.

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