AID	Name	Type	Comment
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 to test synthesized compounds in thallium flux for their ability to dose-dependently inhibit ROMK function.

Experimental methods were as previously described (Lewis, 2009). Briefly, cells were plated in black-walled, clear-bottom plates and treated with Tet overnight to induce the expression of ROMK in serum-free media. The cells were loaded with FluoZin2 dye, incubated for 20 min at RT and washed with assay buffer (0.44 mM NaH2PO4, 4.17 mM NaHCO3, 137.93 mM NaCl, 0.338 mM Na2HPO4, 20 mM HEPES, 0.25 mM K2SO4, adjusted to 343 mOsm with sucrose). The plate was imaged on the Hamamatsu FDSS 6000 system to obtain F0, followed by compound addition. Compounds were serially diluted 3-fold to generate 11-point concentration curves. After 20 min at RT, thallium stimulus buffer (125 mM sodium gluconate, 12 mM thallium sulfate, 1 mM magnesium sulfate, 1.8 mM calcium gluconate, 5 mM glucose, 10 mM HEPES, pH 7.3, adjusted to 343 mOsm with sucrose) was added while simultaneously imaging for a total of 2 min acquisition time.

Data Handling:
The kinetic fluorescence values (F) from each well were divided by the initial frame of the read (F0) to give the static ratio (F/F0) which corrects for variability in cell number and dye loading. The slope of the static ratio from 7 to 12 seconds was calculated for each compound concentration. An automated data analysis pipeline generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org) was used. TPNQ and assay buffer control wells were included on every plate and used to calculate the Z' (Zhang et al., 1999). The data were normalized to % TPNQ activity and the concentration response curves fit using Prism 5.0.

Grant Number: R21 NS057041-01