Manual electrophysiological patch clamp assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
BioAssay Type: Electrophysiology, Patch Clamp, Orthogonal Assay, Specificity Screen, Multiple Concentration Activity in Multiplicates Observed ..more
BioActive Compounds: 10
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center (JHICC_ Kir2.1_inhibitors_ME_2)
BioAssay Type: Electrophysiology, Patch Clamp, Orthogonal Assay, Specificity Screen, Multiple 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: Hao-ran Wang Ph.D., Meng Wu Ph.D., Haibo Yu Ph.D., Bill Shi Ph.D., David Meyers Ph.D., and Jia Xu Ph.D.
Name: Manual electrophysiological patch clamp assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
See the related essay (PubChem AID: 1672).
The purpose of this orthogonal assay is to test the compounds identified in the primary screen and subsequent validation and secondary screens for Kir2.1 (potassium inwardly-rectifying channel J2, KCNJ2) (Pubchem SAID 1843) using manual patch clamp assay for evaluation of SAR compounds.
Protocol for manual patch clamp 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. The electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). Pipette resistance was around 3-4 megaohms.
3. Whole-cell currents of Kir2.1 were recorded by using an Axopatch 200B amplifier. The bath and pipette solution contained 140 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM Hepes (pH 8.5) and 140 mM KCl, 2 mM EDTA, 10 mM Hepes (pH 7.4), respectively. To apply the compound, 7-10ml of compound solution was injected by a 10ml syringe into recording chamber containing 0.5 ml bath solution. Excessive solution was removed by suction.
4. To record Kir2.1 current, voltage was stepped from 0 mV holding potential to -100mV (500 ms) with an interval of 30 seconds. Capacitance and access resistance were monitored and 75% compensated. Currents were filtered at 1 kHz, and data were acquired at 5 kHz with a Digidata 1322A computer interface and pClamp 9.2 software (Axon Instruments).
5. To check the quality of seal during recording, each voltage step was followed by a ramp protocol (500ms, from -100mV to 100 mV). Cells losing seal during recording were identified by a sudden increase of outward current in the ramp protocol, in which case, cell will be dropped.
6. Data were analyzed using pClamp 8 followed by Origin 6. The percentage of inhibition of the tested compounds was calculated with the following formula:
Percentage (%) = (Current(prior cpd)- Current(post cpd))/ Current(prior cpd)*100
Percentage (%): Percentage of current inhibition observed post the application of the test compound.
Current(prior cpd): Current recorded prior the test compound application at -100 mV
Current(post cpd): Current recorded post the test compound application at -100 mV
7. Outcome assignment:
If the test compound causes inhibition of the Kir2.1 current at -100 mV in any concentrations tested, the compound is considered to be active.
If the test compound does not cause inhibition of the Kir2.1 current at -100 mV in any concentrations tested, the compound is designated as inactive.
8. Score assignment:
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)