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BioAssay: AID 1922

High throughput discovery of novel modulators of ROMK K+ channel activity: Selectivity by Patch Clamp

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 more ..
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 1922
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (JD001_selectivity_EP)
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-09-04

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
Related Experiments
AIDNameTypeComment
2436High-throughput Discovery of Novel Modulators of ROMK K+ Channel ActivitySummarydepositor-specified cross reference
2753High throughput discovery of novel modulators of ROMK K+ channel activity: Dose-Response AssayConfirmatorydepositor-specified cross reference
1916High throughput discovery of novel modulators of ROMK K+ channel activity: Analog Dose-ResponseConfirmatorysame project related to Summary assay
1917High throughput discovery of novel modulators of ROMK K+ channel activity: Retest of Primary HitsOthersame project related to Summary assay
1918High throughput discovery of novel modulators of ROMK K+ channel activity: Primary ScreenScreeningsame project related to Summary assay
1924High throughput discovery of novel modulators of ROMK K+ channel activity: Ancillary ActivityOthersame project related to Summary assay
435017High throughput discovery of novel modulators of ROMK K+ channel activity: Analog Library TestingConfirmatorysame project related to Summary assay
Description:
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.
Protocol
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).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ROMK 1 % Block% Inhibition at 120mV for ROMK treated with 1uM of compoundFloat
2ROMK 1 SEMStandard Error of Measure for % Inhibition at 120mV for ROMK treated with 1uM of compoundFloat
3ROMK 1 NNumber of replicates for % Inhibition at 120mV for ROMK treated with 1uM of compoundFloat
4ROMK 1 pp value for % Inhibition at 120mV for ROMK treated with 1uM of compoundString
5ROMK 3 % Block% Inhibition at 120mV for ROMK treated with 3uM of compoundFloat
6ROMK 3 SEMStandard Error of Measure for % Inhibition at 120mV for ROMK treated with 3uM of compoundFloat
7ROMK 3 NNumber of replicates for % Inhibition at 120mV for ROMK treated with 3uM of compoundFloat
8ROMK 3 pp value for % Inhibition at 120mV for ROMK treated with 3uM of compoundString
9ROMK 10 % Block% Inhibition at 120mV for ROMK treated with 10uM of compoundFloat
10ROMK 10 SEMStandard Error of Measure for % Inhibition at 120mV for ROMK treated with 10uM of compoundFloat
11ROMK 10 NNumber of replicates for % Inhibition at 120mV for ROMK treated with 10uM of compoundFloat
12ROMK 10 pp value for % Inhibition at 120mV for ROMK treated with 10uM of compoundString
13ROMK Ba % Block% Inhibition at 120mV for ROMK treated with 2mM of bariumFloat
14ROMK Ba SEMStandard Error of Measure for % Inhibition at 120mV for ROMK treated with 2mM of bariumFloat
15ROMK Ba NNumber of replicates for % Inhibition at 120mV for ROMK treated with 2mM of bariumFloat
16ROMK Ba pp value for % Inhibition at 120mV for ROMK treated with 2mM of bariumString
17Kir2.1 % Block% Inhibition at 120mV for Kir2.1 treated with 10uM of compoundFloat
18Kir2.1 SEMStandard Error of Measure for % Inhibition at 120mV for Kir2.1 treated with 10uM of compoundFloat
19Kir2.1 NNumber of replicates for % Inhibition at 120mV for Kir2.1 treated with 10uM of compoundFloat
20Kir2.1 pp value for % Inhibition at 120mV for Kir2.1 treated with 10uM of compoundString
21Kir2.1 Ba % Block% Inhibition at 120mV for Kir2.1 treated with 2mM of bariumFloat
22Kir2.1 Ba SEMStandard Error of Measure for % Inhibition at 120mV for Kir2.1 treated with 2mM of bariumFloat
23Kir2.1 Ba NNumber of replicates for % Inhibition at 120mV for Kir2.1 treated with 2mM of bariumFloat
24Kir2.1 Ba pp value for % Inhibition at 120mV for Kir2.1 treated with 2mM of bariumString
25Kir4.1 % Block% Inhibition at 120mV for Kir4.1 treated with 10uM of compoundFloat
26Kir4.1 SEMStandard Error of Measure for % Inhibition at 120mV for Kir4.1 treated with 10uM of compoundFloat
27Kir4.1 NNumber of replicates for % Inhibition at 120mV for Kir4.1 treated with 10uM of compoundFloat
28Kir4.1 pp value for % Inhibition at 120mV for Kir4.1 treated with 10uM of compoundString
29Kir4.1 Ba % Block% Inhibition at 120mV for Kir4.1 treated with 2mM of bariumFloat
30Kir4.1 Ba SEMStandard Error of Measure for % Inhibition at 120mV for Kir4.1 treated with 2mM of bariumFloat
31Kir4.1 Ba NNumber of replicates for % Inhibition at 120mV for Kir4.1 treated with 2mM of bariumFloat
32Kir4.1 Ba pp value for % Inhibition at 120mV for Kir4.1 treated with 2mM of bariumString
33Kir7.1 % Block% Inhibition at 120mV for Kir7.1 treated with 10uM of compoundFloat
34Kir7.1 SEMStandard Error of Measure for % Inhibition at 120mV for Kir7.1 treated with 10uM of compoundFloat
35Kir7.1 NNumber of replicates for % Inhibition at 120mV for Kir7.1 treated with 10uM of compoundFloat
36Kir7.1 pp value for % Inhibition at 120mV for Kir7.1 treated with 10uM of compoundString
37Kir7.1 Ba % Block% Inhibition at 120mV for Kir7.1 treated with 2mM of bariumFloat
38Kir7.1 Ba SEMStandard Error of Measure for % Inhibition at 120mV for Kir7.1 treated with 2mM of bariumFloat
39Kir7.1 Ba NNumber of replicates for % Inhibition at 120mV for Kir7.1 treated with 2mM of bariumFloat
40Kir7.1 Ba pp value for % Inhibition at 120mV for Kir7.1 treated with 2mM of bariumString
41Kir2.3 % BLOCK% Inhibition at 120mV for Kir2.3 treated with 10uM of compoundFloat
42Kir2.3 Ba % BLOCK% Inhibition at 120mV for Kir2.3 treated with 2mM of bariumFloat
43Kir2.3 SEMStandard Error of Measure for % Inhibition at 120mV for Kir2.3 treated with 10uM compoundFloat
44Kir2.3 Ba SEMStandard Error of Measure for % Inhibition at 120mV for Kir2.3 treated with 2mM of bariumFloat
45Kir2.3 NNumber of replicates for % Inhibition at 120mV for Kir2.3 treated with 2mM of bariumInteger
46Kir2.3 Ba NNumber of replicates for % Inhibition at 120mV for Kir2.3 treated with 2mM of bariumInteger
47ROMK_geneIDKCNJ1, potassium inwardly-rectifying channel, subfamily J, member 1 String
48Kir2.1_geneIDKCNJ2, potassium inwardly-rectifying channel, subfamily J, member 2String
49Kir2.3_geneIDKCNJ4, potassium inwardly-rectifying channel, subfamily J, member 4String
50Kir4.1_geneIDKCNJ10, potassium inwardly-rectifying channel, subfamily J, member 10String
51Kir7.1_geneIDKCNJ13, potassium inwardly-rectifying channel, subfamily J, member 13 String

RNAi Target.
Additional Information
Grant Number: R21 NS057041-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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