Manual electrophysiological patch clamp assay and ROMK specificity of 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 Compound: 1
Data Source: Johns Hopkins Ion Channel Center
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 and ROMK specificity of 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 AID 1843) using manual patch clamp assay. ROMK (Kir1.1, the renal outer medullary potassium channel) was used as the specificity target.
Protocol for manual patch clamp assay for Kir2.1 and specificity target ROMK:
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 7.4) 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, and were 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)