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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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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
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 ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
2256Summary of efforts to identify compounds that inhibit transient receptor potential cation channel C4 (TRPC4)summary
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)
Classification
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