SAR Analysis for the identification of selective inhibits of the transient receptor potential cation channel C4 (TRPC4): Automated Electrophysiology
Assay Implementation: Jie Shi, Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D. ..more
BioActive Compounds: 13
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Confirmatory, Concentration-Response Relationship 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: Jie Shi, 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): Automated Electrophysiology
The inhibitory effects of test compounds on TRPC4 channels were evaluated in electrophysiologcal assays using a QPatch16 automated electrophysiology instrument. Whole cell recordings were made from HEK293 cells stably expressing TRPC4 channels along with mu-opioid receptors (TRPC4 cells). TRPC4 channels were activated by depolarizing membrane potential ramps and stimulation of GPCR signaling pathways. In control experiments, a mu-opioid agonist, 50 nM DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate) produced robust activation of TRPC4 channels in repeated applications. Following DAMGO application, the effects of test compounds were measured in the continued presence of DAMGO.
The objective of this assay is to validate compounds that inhibit the G-protein receptor coupled activation of the TRPC4 cation channel. This assay employs automated electrophysiology to measure currents in HEK293 cells that express the TRPC4beta and mu-Opioid receptor with voltage clamp protocols in the presence or absence of test compounds.
The Cs-Asp internal solution contained (in mM): 150 Cs-aspartate, 2 MgCl2, 0.36 CaCl2, 1 EGTA, 4 MgATP, 0.3 NaGTP, and 10 HEPES, pH 7.20 with CsOH (calculated free [Ca2+] = 100 nM)
The external solution contained (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 d-glucose, pH 7.40, with NaOH.
1.Cell culture: Cells were 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.TRPC4 cells were grown in to 80-90% confluency and dissociated with trypsin immediately prior to use. Cells were washed, resuspended in external buffer solution at 5-7 million cells per ml and loaded in the QPatch16 instrument. Cells were pipetted to QPlate wells and whole cell recordings established using the instrument's integrated fluidics and pressure controls and following the manufacturer's recommended protocols. Currents were sampled at 5 KHz and filtered at 1 KHz. 80% series resistance compensation was employed.
3.The TRPC4 mu-Opioid expressing HEK293 cells were voltage clamped at 0 mV and brief voltage ramps were applied every 5 s. During the voltage ramp segments, cells are held at 0mV for 200 ms, then at -100 mV for 50ms, followed by a ramp from -100 mV to +120 mV in 110 ms, held at +120 mV for 8 ms, and then back to 0 mV for 180 ms. TRPC4 currents were measured at +120 mV at the end of depolarizing ramp voltage commands. Cells with stable current amplitudes in baseline conditions and following addition of 50 nM DAMGO were used to evaluate effects of test compounds.
4.In order to evaluate effects of test compounds as inhibitors of TRPC4 channels, the channels were first activated by addition of a mu-opioid agonist (50 nM DAMGO) to the extracellular bath solution. Two compound addition protocols were used.
a. Cumulative addition of three doses on individual cells.
Cells were stabilized in saline for 5 min with two saline additions. After two additions of 50 nM DAMGO, cells were recorded for 3 min. Then 1.11uM of compound with the presence of 50 nM DAMGO was added and recorded for 50 sec; second addition of 1.11uM of compound with the presence of 50 nM DAMGO was recorded for another 100 sec. Similar additions and recordings were done with 3.33 and 10 uM of test compounds (with the presence of 50 nM DAMGO) sequentially. Cells are then washed with 2 saline additions for 2.5 min followed by addition of 50 nM DAMGO.
b.Titration experiments using a single dose on individual cells:
Cells were stabilized in saline for 5 min with two saline additions. After two addition of 50 nM DAMGO, cells were recorded for 3 min, followed by another addition of DAMGO for 3 min. One concentration of a compound with 50 nM DAMGO was added twice and recorded for 3 min, followed by another addition for 3 min. Test compound and DAMGO were washed off in saline for 5 min with 3 saline additions. Cells were tested again for their response to 50 nM DAMGO with 2 additions and two 100 sec recording periods. DAMGO was then washed off by two saline additions in 2.5 min.
5.Percent inhibition of TRPC4 currents was calculated at the end of each test period as 100x(1-(Itest-Ibaseline/IDAMGO-Ibaseline)).
6.Percent inhibition values calculated at each test compound concentration for a number of cells were combined and fitted with a Hill equation to provide IC50 values and Hill slope values.
7. Outcome assignment: IC50 values were calculated for each compound using a number of cells in each test plate. If the calculated IC50 values was less that 100 microM, the compound was considered active (Outcome=2). Otherwise, it is designated as inactive (Outcome=1).
8. 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 following criteria: 25 for compounds with 10 microM
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. QPlate16 (Sophion Bioscience, Ballerup, Denmark)
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust, in or on wells of the QPlate plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that modulate the signaling pathways that control TRPC4 channel activation. 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.
* Activity Concentration.
Data Table (Concise)