AID	Name	Type	Comment
2256	Summary of efforts to identify compounds that inhibit transient receptor potential cation channel C4 (TRPC4)	summary

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.

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)

Grant Number: 1 R21 NS056942-01