| Assay Method Information | |
| | GIRK1/4 Activity Assay |
| Description: | The GIRK1/4 activity of a compound according to the present invention was assessed by the following in vitro method.Buffers:a. External buffer: 10 mM NaCl, 50 mM Na Gluconate, 80 mM K Gluconate, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose, pH 7.4; osmolarity 300-310 Osm/L.b. Internal buffer: 30 mM KCl, 100 mM K Gluconate, 1 mM MgCl2, 10 mM HEPES, 1 mM EGTA, 10 mM NaCl, pH 7.2; osmolarity 284-292 Osm/L.Compounds:c. Prepare seven-fold compound dilution series (10 mM to 20 uM) in 100% DMSO, in 384-well polypropylene plates.d. Propafenone (Sigma Aldrich, catalog number P4670) was used as a positive control and DMSO for neutral controle. Resuspend 1 μl of compounds in DMSO into 66.7 μl external buffer in 384-well polypropylene plate and load into Plate 1 section of Molecular Devices Quattro Quattro Setup:f. Load 384-well Population Patch Plate (Molecular Devices #9000-0902) into Quattrog. Fill Quattro F-soak trough with 20% DMSO and 50% EtOHh. Fill Quattro buffer trough with external bufferi. Attach internal buffer flask to Quattro internal buffer tubingj. Attach PBS (Phosphate Buffered Saline, minus Ca++ and Mg++, pH7.4) bottle to F-head & E-head wash on QuattroAntibiotic:k. Resuspend 5.1-5.8 mg amphotericin B (Sigma Aldrich, catalog number A2411) in 175 μl DMSOl. Add the resulting solution to 50 mL internal buffer and attach to antibiotic tubing port on QuattroCells:m. Use GIRK1/4 HEK293 stable cells (obtained from ChanTest, 14656 Neo Parkway, Cleveland, Ohio 44128) grown to 80% confluency in the following cell culture medium: DMEM containing 10% (v/v) Fetal Bovine Serum, Penicillin/Streptomycin (at IX concentration from a 100× stock), 0.5 mg/mL G418 and 0.1 mg/mL Zeocin.n. Detach cells using Detachin (Genlantis, 11011 Torreyana, San Diego, CA 92121), washed with PBS (minus Ca++ and Mg++) and resuspend in external buffer (5 mL final volume at 2.0-2.1×106 cells/mL)o. Load into cell trough of QuattroAssay Protocol:Quattro was controlled using IonWorks v2 software to perform the following steps:p. Added 3.5 μl cells plus 3.5 μl external buffer to wells of Quattro Patch Plate q. Circulated amphotericin B and internal buffer onto cellsr. Applied the following voltage protocol: Pulse 1: 15 mV for 300 milliseconds (ms), followed by Pulse 2: −120 mV for 400 ms, then Pulse 3: −15 mV for 400 ms, and finally Pulse 4: −120 mV to 40 mV over 500 ms (this is a voltage ramp).s. Measured magnitude of inward potassium current at time point between 1200-1220 ms from start of Pulse 1 (i.e., during the voltage ramp phase).t. Added 3.5 μl of diluted compounds (or DMSO) to wells and repeated steps c-d (final compound concentrations are 50 uM to 0.1 uM, and each concentration was tested in quadruplicate i.e., in 4 separate wells).u. The difference between current magnitude pre vs. post-compound gave a measure of GIRK1/4 inhibition. |
| Affinity data for this assay | |
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