Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Front Pharmacol
2011 Nov 30;2:75. doi: 10.3389/fphar.2011.00075.
Show Gene links
Show Anatomy links
Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1.
Raphemot R
,
Lonergan DF
,
Nguyen TT
,
Utley T
,
Lewis LM
,
Kadakia R
,
Weaver CD
,
Gogliotti R
,
Hopkins C
,
Lindsley CW
,
Denton JS
.
???displayArticle.abstract???
The inward rectifier family of potassium (Kir) channels is comprised of at least 16 family members exhibiting broad and often overlapping cellular, tissue, or organ distributions. The discovery of disease-causing mutations in humans and experiments on knockout mice has underscored the importance of Kir channels in physiology and in some cases raised questions about their potential as drug targets. However, the paucity of potent and selective small-molecule modulators targeting specific family members has with few exceptions mired efforts to understand their physiology and assess their therapeutic potential. A growing body of evidence suggests that G protein-coupled inward rectifier K (GIRK) channels of the Kir3.X subfamily may represent novel targets for the treatment of atrial fibrillation. In an effort to expand the molecular pharmacology of GIRK, we performed a thallium (Tl(+)) flux-based high-throughput screen of a Kir1.1 inhibitor library for modulators of GIRK. One compound, termed VU573, exhibited 10-fold selectivity for GIRK over Kir1.1 (IC(50) = 1.9 and 19 μM, respectively) and was therefore selected for further study. In electrophysiological experiments performed on Xenopus laevis oocytes and mammalian cells, VU573 inhibited Kir3.1/3.2 (neuronal GIRK) and Kir3.1/3.4 (cardiac GIRK) channels with equal potency and preferentially inhibited GIRK, Kir2.3, and Kir7.1 over Kir1.1 and Kir2.1.Tl(+) flux assays were established for Kir2.3 and the M125R pore mutant of Kir7.1 to support medicinal chemistry efforts to develop more potent and selective analogs for these channels. The structure-activity relationships of VU573 revealed few analogs with improved potency, however two compounds retained most of their activity toward GIRK and Kir2.3 and lost activity toward Kir7.1. We anticipate that the VU573 series will be useful for exploring the physiology and structure-function relationships of these Kir channels.
Figure 1. VU573 inhibits mGluR8-activated Kir3.1/3.2 channel activity in thallium flux assays. (A) Representative FluoZin-2 fluorescence traces recorded from HEK-293 cells stably expressing mGluR8 and Kir3.1/3.2 before and after co-application of thallium and different doses of glutamate (shaded box). From an 11-point glutamate concentrationâresponse curve (not shown), the glutamate concentration evoking approximately 80% (EC80) of the maximal response (ECmax) was determined and used for subsequent experiments. (B) Representative traces for changes of Tl+-induced FluoZin-2 fluorescence following 20âmin pre-treatment of cells with the indicated concentrations of VU573 and subsequent thallium and glutamate-EC80 addition (shaded box). (C) Meanâ±âSEM concentrationâresponse curve for VU573-dependent inhibition of Kir3.1/3.2 (nâ=â3). The chemical structure of VU573 is shown in the inset. (D) Representative FluoZin-2 fluorescence traces recorded from monoclonal Kir1.1-S44D expressing cells cultured overnight in absence (âTet) or presence (+Tet) of Tetracycline. (E) Representative traces for changes of Tl+-induced FluoZin-2 fluorescence following pre-treatment of cells with the indicated concentrations of VU573 and then exposed to thallium (shaded box). (F) Meanâ±âSEM concentrationâresponse curve for VU573-dependent inhibition of Kir1.1-S44D (nâ=â3).
Figure 2. Effect of VU573 on Kir channels expressed in oocytes. (A) Representative Kir3.1/3.2 current traces recorded from an oocyte using the two-electrode voltage-clamp technique. Oocytes were initially bathed in a potassium-free (0K) solution and then switched to one containing 90 mM potassium (90K) to activate Kir3.1/3.2. After reaching a steady-state, the oocyte was exposed to 10 μM VU573 (in 90K) bath to inhibit Kir3.1/3.2. Residual Kir3.1/3.2 currents were inhibited with 2 mM barium (Ba2+). A final switch back to 0K was used to measure leak current at the end of each experiment. Representative whole-cell current traces recorded from oocytes expressing respectively (B) Kir1.1, (C) Kir2.1, (D) Kir2.3, and (E) Kir7.1 before and after application of 50 μM VU573. Residual Kir currents were inhibited with 2 mM barium (Ba2+). (F) Mean ± SEM percent inhibition of current evoked by Kir3.1/3.2, Kir3.1/3.4, Kir1.1, Kir2.1, Kir2.3, and Kir7.1 with the indicated concentrations of VU573 () or Ba2+ (■) n = 4–6).
Figure 3. VU573-dependent inhibition of Kir3.1/3.2, Kir2.3, and Kir7.1 channel activity expressed in HEK-293 cells. (A,D,G) Representative whole-cell Kir3.1/3.2, Kir2.3, and Kir7.1 current traces recorded from transfected HEK-293 cells. The cell was voltage ramped every 5 s between −120 and 120 mV for 200 ms from a holding potential of −75 mV. Normalized Kir3.1/3.2, Kir2.3, and Kir7.1 currents recorded before (black line) and after reaching steady-state inhibition by 30 μM VU573 (gray line) are shown. (B,E,H) Representative time course traces of VU573-dependent inhibition of Kir3.1/3.2, Kir2.3, and Kir7.1 by 30 μM VU573 using the protocol described above. After achieving whole-cell access, the current was allowed to stabilize before adding 30 μM VU573 and then 2 mM barium (Ba2+) to inhibit channel activity. (C,F,I) Mean ± SEM concentration–response curves for Kir3.1/3.2 (n = 4–7), Kir2.3 (n = 4–6), and Kir7.1 (n = 4–7), respectively.
Figure 4. Development of a thallium flux assay for Kir2.3. (A) Thallium flux-dependent FluoZin-2 fluorescence recorded from monoclonal Kir2.3 T-REx-HEK-293 cells cultured overnight in absence (âTet) or presence (+Tet) of Tetracycline. The fluorescence emission was recorded before and after the addition of extracellular thallium (shaded box). (B) Representative traces for changes of Tl+-induced FluoZin-2 fluorescence following 20âmin pre-treatment of cells with the indicated concentrations of VU573. (C) CRC for VU573-dependent inhibition of Kir2.3 activity. Values are meanâ±âSEM (nâ=â3). A fit of the CRC with a single-site four-parameter logistic function yielded IC50 of 4.7.
Figure 5. Development of a thallium flux assay for Kir7.1 (M125R). (A) Mean ± SEM % inhibition of wild type (closed bars; n = 6–7) or M125R mutant (open bars; n = 4–6) Kir7.1 by the indicated concentration of VU573.Note that the wild type data are reproduced from Figure 3. (B) Thallium flux-dependent FluoZin-2 fluorescence recorded from polyclonalKir7.1 (M125R) T-REx-HEK-293 cells cultured overnight in absence (−Tet) or presence (+Tet) of Tetracycline. The fluorescence emission was recorded before and after the addition of extracellular thallium (shaded box). (C) Representative traces for changes of Tl+-induced FluoZin-2 fluorescence following 20 min pre-treatment of cells with the indicated concentrations of VU573. (D) CRC for VU573-dependent inhibition of Kir2.3 activity. Values are mean ± SEM (n = 3). A fit of the CRC with a single-site four-parameter logistic function yielded IC50 of 4.9 μM.
Figure 6. Lack of effect of R70 on wild type Kir7.1 activity. Average Kir7.1 current traces (n = 4 each) recorded in the absence (A) or presence of (B) 10 μM R70 or (C) 10 μM VU573. (D) Mean ± SEM (n = 4) current–voltage Kir7.1 relationships recorded in the indicated conditions using the voltage-clamp protocol shown in (E).
Bhave,
Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.
2011, Pubmed,
Xenbase
Bhave,
Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.
2011,
Pubmed
,
Xenbase
Bhave,
Small-molecule modulators of inward rectifier K+ channels: recent advances and future possibilities.
2010,
Pubmed
Bridal,
Comparison of human Ether-à-go-go related gene screening assays based on IonWorks Quattro and thallium flux.
2010,
Pubmed
Caballero,
Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification.
2010,
Pubmed
Chuang,
Regulation of IRK3 inward rectifier K+ channel by m1 acetylcholine receptor and intracellular magnesium.
1997,
Pubmed
,
Xenbase
de Boer,
The mammalian K(IR)2.x inward rectifier ion channel family: expression pattern and pathophysiology.
2010,
Pubmed
de Boer,
The anti-protozoal drug pentamidine blocks KIR2.x-mediated inward rectifier current by entering the cytoplasmic pore region of the channel.
2010,
Pubmed
Delpire,
Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2.
2009,
Pubmed
Dobrev,
The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation.
2005,
Pubmed
Ehrlich,
Inward rectifier potassium currents as a target for atrial fibrillation therapy.
2008,
Pubmed
Fallen,
The Kir channel immunoglobulin domain is essential for Kir1.1 (ROMK) thermodynamic stability, trafficking and gating.
2009,
Pubmed
Ferrer,
Carvedilol inhibits Kir2.3 channels by interference with PIP₂-channel interaction.
2011,
Pubmed
Hashimoto,
Tertiapin, a selective IKACh blocker, terminates atrial fibrillation with selective atrial effective refractory period prolongation.
2006,
Pubmed
Hebert,
Molecular diversity and regulation of renal potassium channels.
2005,
Pubmed
Hejtmancik,
Mutations in KCNJ13 cause autosomal-dominant snowflake vitreoretinal degeneration.
2008,
Pubmed
Hibino,
Inwardly rectifying potassium channels: their structure, function, and physiological roles.
2010,
Pubmed
Ho,
Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.
1993,
Pubmed
,
Xenbase
Huang,
Identification of human Ether-à-go-go related gene modulators by three screening platforms in an academic drug-discovery setting.
2010,
Pubmed
Ji,
Rare independent mutations in renal salt handling genes contribute to blood pressure variation.
2008,
Pubmed
Jin,
A novel high-affinity inhibitor for inward-rectifier K+ channels.
1998,
Pubmed
,
Xenbase
Kitamura,
Tertiapin potently and selectively blocks muscarinic K(+) channels in rabbit cardiac myocytes.
2000,
Pubmed
Kobayashi,
Pregnenolone sulfate potentiates the inwardly rectifying K channel Kir2.3.
2009,
Pubmed
,
Xenbase
Krapivinsky,
A novel inward rectifier K+ channel with unique pore properties.
1998,
Pubmed
Leister,
Development of a custom high-throughput preparative liquid chromatography/mass spectrometer platform for the preparative purification and analytical analysis of compound libraries.
2003,
Pubmed
Lewis,
High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1.
2009,
Pubmed
Li,
Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay.
2011,
Pubmed
López-Izquierdo,
The antimalarial drug mefloquine inhibits cardiac inward rectifier K+ channels: evidence for interference in PIP2-channel interaction.
2011,
Pubmed
López-Izquierdo,
Mechanisms for Kir channel inhibition by quinacrine: acute pore block of Kir2.x channels and interference in PIP2 interaction with Kir2.x and Kir6.2 channels.
2011,
Pubmed
Machida,
Effects of a highly selective acetylcholine-activated K+ channel blocker on experimental atrial fibrillation.
2011,
Pubmed
,
Xenbase
Makary,
Differential protein kinase C isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling.
2011,
Pubmed
Matsuda,
Blockade by NIP-142, an antiarrhythmic agent, of carbachol-induced atrial action potential shortening and GIRK1/4 channel.
2006,
Pubmed
Nakamura,
Inwardly rectifying K+ channel Kir7.1 is highly expressed in thyroid follicular cells, intestinal epithelial cells and choroid plexus epithelial cells: implication for a functional coupling with Na+,K+-ATPase.
1999,
Pubmed
Nishida,
Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution.
2002,
Pubmed
Nishida,
Crystal structure of a Kir3.1-prokaryotic Kir channel chimera.
2007,
Pubmed
Niswender,
A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors.
2008,
Pubmed
Ookata,
Localization of inward rectifier potassium channel Kir7.1 in the basolateral membrane of distal nephron and collecting duct.
2000,
Pubmed
Plaster,
Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome.
2001,
Pubmed
,
Xenbase
Ponce-Balbuena,
Tamoxifen inhibits inward rectifier K+ 2.x family of inward rectifier channels by interfering with phosphatidylinositol 4,5-bisphosphate-channel interactions.
2009,
Pubmed
Pondugula,
Glucocorticoid regulation of genes in the amiloride-sensitive sodium transport pathway by semicircular canal duct epithelium of neonatal rat.
2006,
Pubmed
Rodríguez-Menchaca,
The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel.
2008,
Pubmed
Ryan,
Mutations in potassium channel Kir2.6 cause susceptibility to thyrotoxic hypokalemic periodic paralysis.
2010,
Pubmed
Schmalhofer,
A pharmacologically validated, high-capacity, functional thallium flux assay for the human Ether-à-go-go related gene potassium channel.
2010,
Pubmed
Sergouniotis,
Recessive mutations in KCNJ13, encoding an inwardly rectifying potassium channel subunit, cause leber congenital amaurosis.
2011,
Pubmed
Simon,
Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK.
1996,
Pubmed
Tanaka,
A multiple ion channel blocker, NIP-142, for the treatment of atrial fibrillation.
2007,
Pubmed
Tao,
Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 A resolution.
2009,
Pubmed
,
Xenbase
Titus,
A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel.
2009,
Pubmed
van der Heyden,
Finding inward rectifier channel inhibitors: why and how?
2012,
Pubmed
Voigt,
Differential phosphorylation-dependent regulation of constitutively active and muscarinic receptor-activated IK,ACh channels in patients with chronic atrial fibrillation.
2007,
Pubmed
Wakili,
Recent advances in the molecular pathophysiology of atrial fibrillation.
2011,
Pubmed
Walsh,
A real-time screening assay for GIRK1/4 channel blockers.
2010,
Pubmed
Wang,
Selective inhibition of the K(ir)2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR, and pharmacological characterization of ML133.
2011,
Pubmed
Weaver,
A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.
2004,
Pubmed
Welling,
A comprehensive guide to the ROMK potassium channel: form and function in health and disease.
2009,
Pubmed
Yang,
Expression of inwardly rectifying potassium channel subunits in native human retinal pigment epithelium.
2008,
Pubmed
Zaritsky,
The consequences of disrupting cardiac inwardly rectifying K(+) current (I(K1)) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes.
2001,
Pubmed
Zhou,
Primary structure and functional properties of an epithelial K channel.
1994,
Pubmed
,
Xenbase
Zou,
Profiling diverse compounds by flux- and electrophysiology-based primary screens for inhibition of human Ether-à-go-go related gene potassium channels.
2010,
Pubmed
