|Primary cell-based screen for identification of compounds that activate transient receptor potential cation channel C4 (TRPC4). - BioAssay Summary
Assay Implementation: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Kaiping Xu M.S., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
BioActive Compounds: 1510
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Michael Zhu, Ph.D., Ohio State University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS056942-01
Grant Proposal PI: Michael Zhu, Ph.D., Ohio State University
Assay Implementation: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Kaiping Xu M.S., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
HTS execution: Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Kaiping Xu M.S., Meng Wu Ph.D.
Name: Primary cell-based screen for identification of compounds that activate transient receptor potential cation channel C4 (TRPC4).
Transient receptor potential (TRP) channels act as key regulators of many sensory systems including those for thermo-, photo-, and osmosensation [1-3]. A diversity of biological disorders has been linked to TRP channel malfunction ranging from neuropathic pain , cardiovascular diseases , to other diseases involving sensory physiology [6,7].
The TRP family of cation channels can be separated into seven subtypes according to structural similarity. Members of this family are nonselective cation channels that allow entry of extracellular calcium into cells resulting in a depolarization of membrane potential . The mammalian TRPC (canonical) subfamily is proposed to function as store and second-messenger operated cation channels . Disruption of TRPC may cause aberrant modulation of intracellular calcium and changes in membrane potential leading to activation of transcription factors, apoptosis, vascular contractility, platelet activation, and cardiac hypertrophy. Currently there are few pharmacological modulators of the TRPC family and no compounds known to target specific TRPC isoforms .
The objective of the current screen is to identify compounds that increase intracellular Ca2+ levels  within HEK293 cells that express TRPC4 cation channels. Compounds selected as TRPC4 activators will later be counter screened for specificity.
Principle of the assay
To screen for compounds that activate the TRPC4 cation channel, a HEK293 cell line which stably expresses both TRPC4beta and mu-Opioid receptor is employed. Channel activity is monitored by calcium flux with a commercial Fluo4 kit . Compounds that show increase in the Fluo4 fluorescence are considered agonist hits. mu-Opioid receptor activators/potentiators will be excluded through later counter-screening against HEK293 stable cells expressing the mu-Opioid receptor alone.
TRPC4, mu-Opioid receptor, HTS assay, 384, primary, agonist, activator, potentiator, allosteric, FDSS, Calcium, fluorescence, Kinetic, Fluo 4, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
1. Venkatachalam, K., and Montell, C. TRP Channels. Annual Review of Biochemistry 76(1), 387-417 (2007) PMID: 17579562
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3. Reaves, B. J., and Wolstenholme, A. J. The TRP channel superfamily: insights into how structure, protein-lipid interactions and localization influence function. Biochemical Society Transactions 035(1), 77-80 (2007) PMID: 17233605
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5. Vassort G, Alvarez J. Transient receptor potential: a large family of new channels of which several are involved in cardiac arrhythmia. Can J Physiol Pharmacol. 2009 Feb;87(2):100-7.PMID: 19234573
6. Nilius, B. TRP channels in disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1772(8), 805-812 (2007) PMID: 17368864
7. Woudenberg-Vrenken, T. E., Bindels, R. J. M., and Hoenderop, J. G. J. The role of transient receptor potential channels in kidney disease. Nat Rev Nephrol 5(8), 441-449 (2009) PMID: 19546862
8. Zhu, X., Jiang, M., Peyton, M., et al. trp, a Novel Mammalian Gene Family Essential for Agonist-Activated Capacitative Ca2+ Entry. Cell 85(5), 661-671 (1996) PMID: 8646775
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10. Okuhara, D. Y., Hsia, A. Y., and Xie, M. Transient receptor potential channels as drug targets. Expert Opinion on Therapeutic Targets 11(3), 391-401 (2007) PMID: 17298296
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The objective of this assay is to identify compounds that activate the TRPC4 cation channel. A HEK293 cell line which stably expresses both TRPC4beta and mu-Opioid receptor was employed, and channel activity monitored by use of Fluo4, a cell-permeable calcium sensitive dye. Compounds that activate TRC4 result in an increase of intracellular calcium and therefore increase the fluorescence of the dye.
The HEK293 stable cell line was seeded into 384-well plates. After overnight incubation, the cells were loaded with a calcium-sensitive dye, Fluo4, followed by an assay buffer wash. Cell plates were then loaded onto a Hamamatsu FDSS 6000 kinetic imaging plate reader, where compounds were added and real-time fluorescence measured for 110 seconds. Library compound effect was evaluated by computing the integrated ratio of each well which was used for calculation and assignment of a B score for that well (B score Activator Ratio, see Result Definitions, 1). Compounds that effected the fluorescence ratio within two seconds after application were flagged as fluorescent and removed from further analysis (Initial ratio, see Result Definitions, 2). Remaining compounds that scored greater than five times the standard deviation plus the mean of the B score Activator Ratio of all library members were considered to be active as agonists of the TRPC4 cation channel.
Protocol for the TRPC4 project:
1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, 500 ug/mL G418 and 40 ug/mL hygromycin
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of 1x Fluo4 solution to cells
5. Incubate 45 minutes at room temperature (RT) in the dark
6. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), and ECmax of DAMGO
7. Remove Fluo4 dye solution and add 40 ul/well of assay buffer to cells
8. Remove 40 ul solution and add 20 ul/well of assay buffer to cells
9. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 5 seconds at 1Hz to establish baseline
11. Add 4 ul of 7.5x compound stock into the cell plates.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors 
14. Calculate B scores  for test compounds using integrated ratios calculated in Step 12
15. Outcome assignment: If the B score of the test compound is more than 5 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds (B score Activator Ratio>5*SD), AND the ratio of initial fluorescence intensity is within 5 times the standard deviation plus the mean of the ratios of the library compounds, the compound is designated in the Outcome as active (value=2) as an activator of the TRPC4 channel. Otherwise, it is designated as inactive (value=1).
16. Score assignment: An active test compound is assigned a score between 1 and 100 by calculation of Integer((Log10(abs([B score Activator Ratio]))-1.62)*100); they are normalized to the smallest and largest LOG(B score Activator Ratio), B score Activator Ratio, as in the result definition. The inactive test compounds are assigned a score of 0.
List of reagents
1. TRPC4 and mu-Opioid Receptor-expressing HEK293 Cells (provided by Assay Provider Michael Zhu, Ohio State University)
2. PBS: pH7.4 (Invitrogen Cat#10010023)
3. Medium: Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Sigma D5796-500ML)
4. Heat Inactivated Fetal Bovine Serum (Sigma, Cat# F2442)
5. L-Glutamine (Invitrogen, Cat#25030081)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. CellStripper (Mediatech 25-056-Cl)
8. G418: (Invitrogen, Cat#11811-031)
9. Hygromycin#(Mediatech, Cat#30-240-CR)
10. HEPES (Sigma, Cat#H4034)
11. 10XHBSS (#Invitrogen Cat#14065056)
12. Pluronic F-127 (20% solution in DMSO) (Invitrogen Cat#P3000MP)
13. [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO) (Sigma Cat#E7384-10mg10MG)
14. Fluo-4 Calcium Assay Kit (Invitrogen, Cat # F14202)
15. Triple-layer flask (VWR, Cat #62407-082)
16. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #8281903)
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust, in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary, based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)