KCNK9 specificity screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
Assay Implementation: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Haibo Yu Ph.D., Beiyan Zou Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
Sequence: potassium channel, subfamily K, member 9 [Homo sapiens]
More BioActivity Data..
BioActive Compounds: 13
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Specificity Screen, Duplicate, Single 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: 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: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Haibo Yu Ph.D., Beiyan Zou Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
Name: KCNK9 specificity screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
See the related essay (PubChem AID: 1672).
The purpose of this assay is to test the compounds that identified in the primary screen assay (PubChem AID 1672) for their non-specific effects on KCNK9 (KCNK9 potassium channel, subfamily K, member 9 [Homo sapiens]) expressed in HEK293 cells. It employs the same experimental conditions as that of the primary assay, except for the usage of a HEK293 cell line expressing KCNK9 under an inducible promoter. Compounds were tested in duplicates and their effects were evaluated by the calculated FluxOR fluorescence ratio percentage normalized with that from negative controls measured in the absence of compounds. If the compound causes 3SD (of negative controls) or more activity decrease in both duplicates, the compound is considered to be active that might have non-specific effects on the KCNK9-expressing cells. It is NOT specific to inward rectifying potassium ion channel Kir2.1.
Protocol for KCNK9 specificity screen of the Kir2.1 project:
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. Cell plating: Add 50 ul/well of 120,000 cells/ml re-suspended in DMEM/F12 medium with 10% FBS, with tetracycline inducer.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 25 ul/well of 1x FluxOR solution to cells
5. Incubate 90 minutes, at room temperature (RT), in the dark
6. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (IC0), and IC100 of Chlorpromaizne (all with DMSO concentrations matched to that of test compounds)
7. Remove FluxOR dye solution and add 20 ul/well of assay buffer to cells
8. Add 4 ul of 7.5x compound stock into the cell plates via Cybi-Well system
9. Incubate all cell plates for 20 minutes at RT in the dark
10. Prepare 5x stimulus buffer containing 25 mM K2SO4 and 7 mM Tl2SO4
11. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
12. Measure fluorescence for 10 seconds at 1Hz to establish baseline
13. Add 6 ul/well of stimulus buffer onto cells and continue measuring fluorescence for 110 seconds
14. Calculate ratio readout as F(max-min)/F0
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z# factors.
16. Calculate the percentages of tested compounds with the following formula:
Percentage (%) = (Ratio(cpd)- Ratio(Blank))/(Ratio(NC) - Ratio(Blank))*100
Percentage (%): Percentage of the test compound readout over those of negative controls at a concentration of 10 uM
Ratio(cpd): Ratio of the test compound
Ratio(Blank): Ratio of the blank control without stimulus buffer
Ratio(NC): Ratio of the negative controls with stimulus buffer
17. Outcome assignment:
If the compound causes 3SD (of negative controls) or more Percentage (%) decrease AND the fluorescence readout of the compound is within 3SD of the fluorescence of the negative controls, the compound is considered to be active.
If the compound is active in both duplicates, the compound is confirmed as active as a non-specific modulator for KCNK9 in the Outcome. Otherwise, it is designated as inactive.
18. Score assignment:
An inactive test compound is assigned the score of 0.
An active test compound is assigned a score between 15 and 100 by calculation of Integer(Abs((80-[AvRatioPercentage])*1.66)), where AvRatioPercentage is the average of the duplicates of the test compound.
List of reagents
1. HEK-293 cells expressing KCNK9 under inducible promoter (provided by Dr. Sojin Shikano, Dept of Biochemistry & Molecular Genetics, University of Illinois at Chicago)
2. Others (See PubChem AID 1672)
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals, or dust in or on wells of the microtiter plate, or compounds that quench or emit light or fluorescence within the well. 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. The condition was optimized for screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1, NOT for testing modulators of KCNK9. Normalization algorithm is specific for this set of data and cannot be used for comparison with other screens.
** Test Concentration.
Data Table (Concise)