This work was supported by funding from National Institutes of Health grants 1R21NS073097-01 and 1R01DK082884 (J.S.D.) and Foundation for the National Institutes grant PIER11VCTR.

1. Generation of Stable Polyclonal Cell Lines
<ol>
	<li>The establishment of a high quality stable cell line expressing the Kir channel of interest is an important first step toward developing a robust high-throughput screening assay. Constitutive K+ channel overexpression can lead to activation of cell death pathways, stable cell line degeneration and loss of assay performance. To avoid these potential problems and provide a convenient internal control for assay development (see below), a tetracycline-ind...

<ol>
	<li>Ehrlich, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inward+rectifier+potassium+currents+as+a+target+for+atrial+fibrillation+therapy.">Inward rectifier potassium currents as a target for atrial fibrillation therapy.</a> J. Cardiovasc. Pharmacol. 52 (2), 129 (2008).</li><li>Bhave, G., Lonergan, D., Chauder, B. A., Denton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+modulators+of+inward+rectifier+K++channels:+recent+advances+and+future+possibilities.">Small-molecule modulators of inward rectifier K+ channels: recent advances and future possibilities.</a> Future Med. Chem. 2 (5), 757 (2010).</li><li>Swartz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarantula+toxins+interacting+with+voltage+sensors+in+potassium+channels.">Tarantula toxins interacting with voltage sensors in potassium channels.</a> Toxicon. 49 (2), 213 (2007).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-affinity+inhibitor+for+inward-rectifier+K++channels.">A novel high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 37 (38), 13291 (1998).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+stable+form+of+tertiapin:+a+high-affinity+inhibitor+for+inward-rectifier+K++channels.">Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 38 (43), 14286 (1999).</li><li>Roy, A., McDonald, P. R., Sittampalam, S., Chaguturu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+high+throughput+drug+discovery+in+the+public+domain:+a+Mount+Everest+in+the+making.">Open access high throughput drug discovery in the public domain: a Mount Everest in the making.</a> Curr. Pharm. Biotechnol. 11 (7), 764 (2010).</li><li>Weaver, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thallium-sensitive,+fluorescence-based+assay+for+detecting+and+characterizing+potassium+channel+modulators+in+mammalian+cells.">A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.</a> J. Biomol. Screen. 9 (8), 671 (2004).</li><li>Lewis, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+reveals+a+small-molecule+inhibitor+of+the+renal+outer+medullary+potassium+channel+and.">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and.</a> 76 (5), 1094 (2009).</li><li>Bhave, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+selective+small-molecule+inhibitor+of+Kir1.1,+the+renal+outer+medullary+potassium+channel.">Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.</a> Mol. Pharmacol. 79 (1), 42 (2011).</li><li>Raphemot, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Front Pharmacol. 2, 75 (2012).</li><li>Niswender, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Mol. Pharmacol. 73 (4), 1213 (2008).</li></ol>

Data treatment: Once the data are collected, a common step in the analysis involves normalizing each well's fluorescence response, F, to its initial value at the beginning of the experiment, F0. This is commonly referred to as the "static ratio" and symbolized "F/F0". In cases where F0 is dominated by the indicator dye the static ratio operation will substantially correct for many factors such as disuniformities in illumination, signal collection, an...

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.
The formula in step 7.1 of the Protocol has been updated from:
Z prime = 1- (3SDp + 3SDn)/|meanp + meann |
to:
Z prime = 1- (3SDp + 3SDn)/|meanp - meann |

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.

Erratum: High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>pcDNA5/TO</td>
 <td>Invitrogen</td>
 <td>V1033-20</td>
 <td>Tetracycline-inducible expression vector</td>
 </tr>
 <tr>
 <td>T-REx-HEK293 cells</td>
 <td>Invitrogen</td>
 <td>R71007</td>
 <td>Tetracycline-inducible cell line</td>
 </tr>
 <tr>
 <td>Lipofectamine LTX/Plus Reagent</td>
 <td>Invitrogen</td>
 <td>15338100</td>
 <td>Transfection reagent</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>ATLANTA Biologicals</td>
 <td>S11550</td>
 <td>Cell culture media</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965</td>
 <td>Cell culture media</td>
 </tr>
 <tr>
 <td>Hygromycin B</td>
 <td>Invitrogen</td>
 <td>10687-010</td>
 <td>Cell culture media</td>
 </tr>
 <tr>
 <td>Blasticidin S</td>
 <td>Invitrogen</td>
 <td>R210-01</td>
 <td>Cell culture media</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>Cell culture media</td>
 </tr>
 <tr>
 <td>HBSS-divalent free</td>
 <td>Mediatech</td>
 <td>21022CV</td>
 <td>Cell washing</td>
 </tr>
 <tr>
 <td>Trypsin-0.25%</td>
 <td>Mediatech</td>
 <td>25053CI</td>
 <td>Cell dissociation</td>
 </tr>
 <tr>
 <td>Tetracycline-HCl</td>
 <td>Sigma</td>
 <td>T9823</td>
 <td>Induction reagent</td>
 </tr>
 <tr>
 <td>Dialyzed FBS</td>
 <td>ATLANTA Biologicals</td>
 <td>S12650</td>
 <td>Plating media</td>
 </tr>
 <tr>
 <td>FluoZin-2</td>
 <td>Invitrogen</td>
 <td>F24189</td>
 <td>Fluorescent dye</td>
 </tr>
 <tr>
 <td>Pluronic F-127</td>
 <td>Invitrogen</td>
 <td>P-3000MP</td>
 <td>Dye loading</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Invitrogen</td>
 <td>14175</td>
 <td>Assay buffer</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td>Assay buffer</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Sigma</td>
 <td>S6297</td>
 <td>Tl+ stimulus buffer</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Sigma</td>
 <td>M2643</td>
 <td>Tl+ stimulus buffer</td>
 </tr>
 <tr>
 <td>CaSO4&bull;2H2O</td>
 <td>Sigma</td>
 <td>C3771</td>
 <td>Tl+ stimulus buffer</td>
 </tr>
 <tr>
 <td>D-Glucose</td>
 <td>Sigma</td>
 <td>G7528</td>
 <td>Tl+ stimulus buffer</td>
 </tr>
 <tr>
 <td>Thallium sulfate</td>
 <td>Aldrich</td>
 <td>204625</td>
 <td>Tl+ stimulus buffer</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H4034</td>
 <td>Tl+ stimulus buffer</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D4540</td>
 <td>Solvent</td>
 </tr>
 <tr>
 <td>Eight-channel electronic pipettor</td>
 <td>Biohit</td>
 <td>E300</td>
 <td>Cell plating in 384-well plates</td>
 </tr>
 <tr>
 <td>BD PureCoat amine-coated 384-well plates</td>
 <td>BD Biosciences</td>
 <td>356719</td>
 <td>Assay microplates</td>
 </tr>
 <tr>
 <td>Echo qualified 384-Well polypropylene microplate (384PP)</td>
 <td>Labcyte</td>
 <td>P-05525</td>
 <td>Compound source microplates</td>
 </tr>
 <tr>
 <td>384-well polypropylene microplates</td>
 <td>Greiner Bio-One</td>
 <td>781280</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Multidrop Combi reagent dispenser</td>
 <td>Thermo Scientific</td>
 <td>5840300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ELx405 microplate washer</td>
 <td>BioTek</td>
 <td>ELx405HT</td>
 <td>Automated cell washing</td>
 </tr>
 <tr>
 <td>Echo liquid handler</td>
 <td>Labcyte</td>
 <td>Labcyte Echo 550</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bravo automated liquid handling platform</td>
 <td>Agilent Technologies</td>
 <td>Standard model</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hamamatsu FDSS 6000</td>
 <td>Hamamatsu</td>
 <td>&nbsp;</td>
 <td>Kinetic imaging plate reader</td>
 </tr>
 </tbody>
</table>
Table 1. List of Materials and Reagents.

high-throughput screening for small-molecule modulators of inward rectifier potassium channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

The use of a tetracycline-inducible expression system provides a convenient internal control for distinguishing Tl+ flux through endogenous pathways and the Kir channel of interest. Figure 1 shows some examples of cell plating maps used in different types of experiments. The positions of wells containing uninduced or tetracycline-induced cells are indicated with different colors. Figure 2A shows the source plate map used to determine the optimal Tl+ concentration fo...

Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

high-throughput-screening-for-small-molecule-modulators-inward

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Biology

14.9K visualizações.  Vanderbilt University School of Medicine. Os métodos para o desenvolvimento e validação de um ensaio quantitativo de fluorescência para medir a actividade de potássio rectificador de entrada (Kir) canais de rastreio de alto rendimento composto é apresentado.