Abstract
The renal outer medullary potassium channel (ROMK) is expressed in the kidney tubule and critically regulates sodium and potassium balance. The physiological functions of other inward rectifying K+ (Kir) channels expressed in the nephron, such as Kir7.1, are less well understood in part due to the lack of selective pharmacological probes targeting inward rectifiers. In an effort to identify Kir channel probes, we performed a fluorescence-based, high-throughput screen (HTS) of 126,009 small molecules for modulators of ROMK function. Several antagonists were identified in the screen. One compound, termed VU590, inhibits ROMK with submicromolar affinity, but has no effect on Kir2.1 or Kir4.1. Low micromolar concentrations inhibit Kir7.1, making VU590 the first small-molecule inhibitor of Kir7.1. Structure-activity relationships of VU590 were defined using small-scale parallel synthesis. Electrophysiological analysis indicates that VU590 is an intracellular pore blocker. VU590 and other compounds identified by HTS will be instrumental in defining Kir channel structure, physiology, and therapeutic potential.
- ROMK, renal outer medullary K+ channel
- Kir, inward rectifying potassium
- EK, potassium equilibrium potential
- VU590, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane
- HTS, high-throughput screen
- CCD, cortical collecting duct
- HEK, human embryonic kidney
- TPNQ, Tertiapin Q
- PBS, phosphate-buffered saline
- TBST, Tris-buffered saline/Tween 20
- DMSO, dimethyl sulfoxide
- DCM, dichloromethane
- Tet, tetracycline
- RMP, resting membrane potential
- CRC, concentration-response curve
- FDSS, Functional Drug Screening System 6000.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant 1R21-NS57041-1]; the National Institutes of Health National Institute of Mental Health Molecular Libraries Screening Centers Network [Grants U54-MH074427, 1U54-MH084659-01]; and a National Kidney Foundation Postdoctoral Fellowship grant.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.059840
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ABBREVIATIONS:
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L.M.L. and G.B. contributed equally to this work.
- Received July 27, 2009.
- Accepted August 25, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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