SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9): Manual Electrophysiology
The purpose of this assay is to evaluate compound inhibition of KCNK9 two pore domain potassium channels using conventional voltage clamp technique. Whole cell recordings were made from HEK293 cells stably expressing KCNK9 channels. ..more
BioActive Compound: 1
Depositor Specified Assays
Principle of the assay
The purpose of this assay is to evaluate compound inhibition of KCNK9 two pore domain potassium channels using conventional voltage clamp technique. Whole cell recordings were made from HEK293 cells stably expressing KCNK9 channels.
1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Fetal Bovine Serum (HiFBS), 15 microg/mL Blasticidin S and 400 microg/mL hygromycin
2. The stable HEK 293 cell line expressing inducible KCNK9 expression was seeded on to poly-D-lysine coated cover slips two days before testing and induced with 1microg/ml tetracycline 16-24h prior to recording.
3. Recording pipettes are pulled with borosilicate glass (World Precision Instruments Inc, Sarasota, FL) to 2-5 M ohm when filled with a pipette solution containing 140 mM KCl, 1 mM MgCl2, 5 mM EGTA, 5 mM HEPES, 1 mM MgATP, pH 7.3 and placed in the bath solution containing 140 mM NaCl, 5 mM KCl, 0.5 mM CaCl2,1 mM MgCl2, and 10 mM HEPES, pH adjusted to 7.3 with NaOH. Isolated cells are voltage-clamped in whole-cell mode with an AxonPatch 200B amplifier (Axon Instruments Inc, Union City, CA), and currents recorded at 5 kHz. KCNK9 currents were measured by voltage ramps from -120 mV to 40 mV applied every 30 seconds. The patched cells are continuously perfused with bath solutions through a gravity-driven perfusion system.
4. Percent inhibition of KCNK9 currents was calculated as 100x (I5k-Itest)/(I5k) where I is the measured current at -20 mV.
5. Outcome assignment
If the test compound causes greater than 50% inhibition of KCNK9 at 1 microM, the compound is considered to be active (Outcome=2). Otherwise, the compound is designated as inactive (Outcome=1).
6. 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 0
A. Percent inhibition at 1 microM.
B. Standard deviation of percent inhibition values.
C. Number of cells included in each determination.
** Test Concentration.
Data Table (Concise)