Secondary automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
BioAssay Type: Automated Electrophysiology, Patch Clamp, Single Concentration Activity in Multiplicates Observed ..more
BioActive Compounds: 227
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_inhibitors_IWS)
BioAssay Type: Automated Electrophysiology, Patch Clamp, Single Concentration Activity in Multiplicates 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: Elena Makhina Ph.D., University of Pittsburgh
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA026212-01
Grant Proposal PI: Elena Makhina Ph.D., University of Pittsburgh
Assay Implementation: Haibo Yu Ph.D., Hao-ran Wang Ph.D., Owen McManus Ph.D., and Meng Wu Ph.D.
Name: Secondary automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
See the related assay (PubChem AID: 1672).
The purpose of this assay is to test the compounds generated from the primary screen and subsequent validation and secondary screens for Kir2.1 (potassium inwardly-rectifying channel J2, KCNJ2) (Pubchem SAID 1843) using automated population patch clamp electrophysiology system IonWorks (Molecular Devices).
Protocol for automated electrophysiology assay for Kir2.1:
1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50ug/ml streptomycin, and 500ug/ml G418.
2. Prepare 3X compound plates: test compounds are prepared using external buffer (140 mM K-gluconate, 1.8 mM CaCl2,1 mM MgCl2, 10 mM HEPES, 10 mM Glucose, pH 7.4); controls are external buffer (both with DMSO concentrations matched to that of test compounds)
3. Cell preparation: Cells are dislodged using 0.125 % Trypsin/EDTA and resuspended at 2X10E6 /mL in external buffer.
4. Patchplate hole test on Ionworks: External buffer is added to each well of a 384-well PPC plate, 3.5 uL/well, and hole resistance of each well is measured.
5. Cell plating on Ionworks: Cells are dispensed to the 384-well PPC plate on Ionworks, 3.5 uL/well.
6. Membrane permeablization: Cells are exposed to 0.1mg/mL amphotericin B in internal buffer (40 mM KCl, 100 mM K-gluconate, 1 mM MgCl2, 5mM HEPES,2 mM CaCl2 pH 7.2).
7. Currents are measured using the following voltage protocol: Hold cells at +10 mV, depolarize to -100 mV for 800 ms, back to +10 mV for 600 ms and run a ramp from -100 mV to +100 mV in 800 ms before back to +10 mV.
8. Dispense 3.5 ul /well of compounds to each well on Ionworks and incubate for 4 minutes.
9. Measure currents using the same protocol described in 7.
10. Calculate Inhibition (%) = (Iprecompound -Ipostcompound)/Iprecompound*100%
11. Calculate the average and standard deviation for negative control in each plate
12. Calculate the percentage with the following formula:
Percentage = Inhibition (%) - (Avg-3*SD)
Where: Percentage: percentage change of compound over those of negative controls and 3 times of its standard deviation.
Avg: average of potentiation (%) of those of negative controls.
SD: standard deviation of potentiation (%) of those of negative controls.
13. Outcome assignment:
If the test compound causes Percentage of the Kir2.1 current at -100 mV in concentration tested to be greated than 0, the compound is considered to be active. Otherwise, the compound is designated as inactive.
14. Score assignment:
An active test compound is assigned between 5 and 100 by Int (([Percentage]+4.54)/88.2)*0.95*100.
An inactive test compound is assigned the score of 0.
** Test Concentration.
Data Table (Concise)