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BioAssay: AID 492983

SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Manual Electrophysiology

Assay Implementation: Michael Zhu, Ph.D., Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D. ..more
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AID: 492983
Data Source: Johns Hopkins Ion Channel Center (TRPC4_inh_ManualEP)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-12-14
Hold-until Date: 2011-06-15
Modify Date: 2011-06-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
2256Summary of efforts to identify compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Summary3 depositor-specified cross reference
2247Primary cell-based screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4).Screening same project related to Summary assay
2636Confirmatory screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
2637Second confirmatory screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
434942Confirmation dose response assay for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Confirmatory same project related to Summary assay
434948Specificity screen against TRPC6 for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
434978Second counter screen for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
492981SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4) in TRPC6 expressing cells: MPD AssayScreening same project related to Summary assay
492987SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4) in TRPC6 expressing cells: MPD Assay CRCConfirmatory same project related to Summary assay
492988SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Primary HTS assayScreening same project related to Summary assay
492991SAR Analysis for the identification of selective inhibits of the transient receptor potential cation channel C4 (TRPC4): Automated ElectrophysiologyConfirmatory same project related to Summary assay
492994SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Primary HTS assay CRCConfirmatory same project related to Summary assay
492995SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): TRPC4 Serotonin Receptor CRC MPD assayConfirmatory same project related to Summary assay
492996SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): ACh assay CRCConfirmatory same project related to Summary assay
504554SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C6 (TRPC6) in cells expressing TRPC4: C4 assay CRCConfirmatory same project related to Summary assay
504589SAR Analysis for the identification of selective inhibits of the transient receptor potential cation channel C4 (TRPC4): Automated Electrophysiology_2Confirmatory same project related to Summary assay
504866SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C6 (TRPC6) in cells expressing TRPC4: C4 assay CRC 2Confirmatory same project related to Summary assay
Description:
Data Source: Ohio State University
BioAssay Type: other, 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: Michael Zhu, Ph.D., Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D.

Name: SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Manual Electrophysiology

Assay overview:

The objective of this assay is to validate compounds that inhibit the G-protein coupled receptor activation of the TRPC4 cation channel. This assay employs manual electrophysiology to investigate the currents recorded from HEK cells that express the TRPC4beta and mu-Opioid receptor through voltage clamp protocols in the presence or absence of test compounds.
Protocol
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. Electrophysiological recording: For whole cell patch clamp recordings, the internal solution has ~400 nM free Ca and is composed of (in mM): 110 CsCl, 10 HEPES, 10 BAPTA 1 MgCl2, 6.46 CaCl2, pH adjusted to 7.2 with HCl. The osmolarity is 274. The external solution contains (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH7.4 adjusted with NaOH. The pipette resistance is 2-4 Mohms and recording is performed at the room temperature (22C). Cells were held at 0 mV and repeated voltage commands stepping to -100 mV for 20 ms followed by a voltage ramp to 100 mV in 100 ms were given at a 0.5 sec interval.
3. Test compound application: Cells were pretreated with or without 1 microM of drug for > 2 min before the addition of 10 microM carbachol + 0.1 microM DAMGO. This condition was used to maximally activate TRPC4 whole-cell currents
4. Current amplitudes were measured at -100 mV and +100 mV for each cell and normalized to cell capacitance. Current densities were compared between treated and untreated cells.
5. Outcome assignment: If the average current density for treated cells was decreased by >40 % at either voltage, the compound was considered active (Outcome=2). Otherwise, it is designated as inactive (Outcome=1).
6. Score assignment: an inactive test compound is assigned the score of 0. An active test compound is assigned a score between 1 and 100 according to the maximal percent block at either voltage.
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. [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO) (Sigma Cat#E7384-10mg10MG)
11. Carbamoylcholine chloride (carbachol) (Sigma Cat#C2409-25)
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Percent inhibition at -100 mV (1 uM)Percent inhibition at -100 mV (1 uM)Float
2Percent inhibition at +100 mV (1 uM)Percent inhibition at +100 mV (1 uM)Float
3Control average current at -100 mV(pA/pF)Control average current at -100 mV(pA/pF)Float
4Control SEM at -100 mV (pA/pF)Control SEM at -100 mV (pA/pF)Float
5Control average current at +100 mV(pA/pF)Control average current at +100 mV(pA/pF)Float
6Control SEM at +100 mV (pA/pF)Control SEM at +100 mV (pA/pF)Float
7Control number of cellsControl number of cellsInteger
8Average current (pA/pF) at -100 mV in 1 uMAverage current (pA/pF) at -100 mV in 1 uM compoundFloat
9Current SEM (pA/pF) at -100 mV in 1 uMCurrent SEM (pA/pF) at -100 mV in 1 uM compoundFloat
10Average current (pA/pF) at +100 mV in 1 uMAverage current (pA/pF) at +100 mV in 1 uM compoundFloat
11Current SEM (pA/pF) at +100 mV in 1 uMCurrent SEM (pA/pF) at +100 mV in 1 uM compoundFloat
12Number of cells in 1 uMNumber of cells in 1 uM compoundInteger
Additional Information
Grant Number: 1 R21 NS056942-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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