AID	Name	Type	Comment
2753	High throughput discovery of novel modulators of ROMK K+ channel activity: Dose-Response Assay	confirmatory
2436	High-throughput Discovery of Novel Modulators of ROMK K+ Channel Activity	summary

Assay Provider: Jerod Denton
Assay Provider Affiliation: Vanderbilt University
Grant Title: High throughput discovery of novel modulators of ROMK K+ channel activity
Grant Number: R21 NS057041-01

The Renal Outer Medullary Potassium channel (ROMK, Kir1.1) is expressed in the renal tubule where it critically regulates fluid and electrolyte homeostasis (Hebert, 2005). An emerging body of evidence suggests that ROMK could be a target for a novel class loop diuretic that lowers blood pressure while preserving plasma potassium levels (Ji, 2008). Furthermore, homozygous loss-of-function mutations in the gene encoding ROMK (KCNJ1) cause antenatal Bartter syndrome, a severe salt and water wasting disease in infants (Simon, 1996). ROMK is thus an important pharmacological target for the management of disease. Its actual therapeutic value and drugability, however, are unknown due to the lack of small-molecule probes targeting the channel. The discovery of ROMK modulators will provide important new tools for studying the structure, function and therapeutic potential of ROMK and other inward rectifying potassium channels.

The purpose of this assay was to use patch-clamp electrophysiology to test lead compounds for inhibition of potassium inward rectifier (KIR) family members (ROMK/KIR1.1, KIR2.1, KIR2.3, KIR4.1, and KIR7.1).

Experiments were done as previously described (Lewis, 2009). Cells containing ROMK/KIR1.1 were cultured in the presence of Tet the night before patch clamp experiments and HEK-293 cells were transiently transfected with 0.5-1.0 ug of channel expression vector and 0.5 ug of pcDNA3.1-EGFP (transfection marker). Experiments were performed approximately 24 h post-transfection by rinsing the cells with divalent-free buffer (145 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 0.3 mM Na2HP04, 0.3 mM KH2PO4, 5.6 mM D-glucose, pH 7.4, 290 mOsm), dissociating by brief exposure to 0.25% Trypsin containing 1 mM EDTA, plating on poly-L-lysine-coated round glass coverslips and allowing to recover at 37 degrees C in a 5% CO2 incubator for at least 1 h. The coverslips were moved to a small-volume perfusion chamber (Warner Instruments, Hamden, CT) and mounted on the stage of a Nikon Eclipse TE2000-U inverted microscope. Patch electrodes were pulled from silanized 1.5 mm outer diameter borosilicate microhematocrit tubes with a Narishige PP-830 two-stage puller. Electrode resistance ranged from 2.5 to 3.5 MOhm when filled with the following intracellular solution (in mM) 135 KCl, 2 MgCl2, 1 EGTA, 10 HEPES free acid, 2 Na2ATP (Roche, Indianapolis, IN), pH 7.3, 275 mOsm. The standard bath solution contained (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, 10 HEPES free acid, pH 7.4, 290 mOsm. Whole-cell currents were recorded under voltage-clamp conditions using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The cell membrane potential was recorded in current clamp mode. Electrical connections to the amplifier were made using Ag/AgCl wires and 3 M KCl/agar bridges. Electrophysiological data were collected at 5 kHz and filtered at 1 kHz. Data acquisition and analysis were performed using pClamp 9.2 software (Axon Instruments).

Grant Number: R21 NS057041-01