Name: a fluorescent screening assay for identifying modulators of girk channels
Uploaded: 2012-04-24T13:00:00
Description: Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

Dette arbejde blev støttet af US Public Health Service pris NS-071.530.

1. Fremstillinq af celler <ol><li> Vokser hypofyse AtT20 celler i Dulbeccos modificerede Eagles medium (DMEM) suppleret med 10% hesteserum og opretholde kulturer ved 37 ° C i en befugtet atmosfære af 5% CO2. </li><li> Fastholde den ved subkultivering cellerne hver 5 til 7 dage under anvendelse af en standard trypsinisering procedure. </li><li> Coate brøndene i sorte, klarbundede, 96-brønds plader med 50 ul af poly-L-lysin. Tillader brøndene tørre i 30 minutter i inkubatoren. </li><li> Pladen cellerne ...

<ol>
	<li>Hibino, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inward+rectifying+potassium+channels:+their+structure,+function+and+physiological+roles.">Inward rectifying potassium channels: their structure, function and physiological roles.</a> Physiol. Rev. 90, 291-366 (2010).</li><li>Lusscher, C., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+for+G+protein-gated+inwardly+rectifying+potassium+(GIRK)+channels+in+health+and+disease.">Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease.</a> Nat. Rev. Neurosci. 11, 301-315 (2010).</li><li>Kobayashi, T., Ikeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-activated+inwardly+rectifying+potassium+channels+as+potential+therapeutic+targets.">G protein-activated inwardly rectifying potassium channels as potential therapeutic targets.</a> Cur. Pharm. Des. 12, 4513-4523 (2006).</li><li>Terstappen, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+native+and+recombinant+ion+channels+using+a+high-capacity+nonradioactive+rubidium+efflux+assay.">Functional analysis of native and recombinant ion channels using a high-capacity nonradioactive rubidium efflux assay.</a> Anal. Biochem. 272, 149-155 (1999).</li><li>Cheng, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+HERG+potassium+channel+function+assay:+an+old+assay+with+a+new+look.">A high-throughput HERG potassium channel function assay: an old assay with a new look.</a> Drug Dev. Ind. Pharm. 28, 177-191 (2002).</li><li>Zhang, J. -. H., Chung, T. D. Y., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> J. Biomol. Screen. 4, 67-73 (1999).</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> Biochem. 37, 13291-13299 (1998).</li><li>Inomata, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-arrhythmic+agents+act+differently+on+the+activation+phase+of+the+Ach-response+in+guinea+pig+atrial+myocytes.">Anti-arrhythmic agents act differently on the activation phase of the Ach-response in guinea pig atrial myocytes.</a> Br. J. Pharmacol. 108, 111-116 (1993).</li><li>Tang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+high+throughput+functional+assay+methods+for+HERG+potassium+channel.">Development and evaluation of high throughput functional assay methods for HERG potassium channel.</a> J. Biomol. Screen. 6, 325-331 (2001).</li><li>Wolff, C., Fuks, B., Chatelain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+membrane+potential-sensitive+fluorescent+probes+and+their+use+in+ion+channel+screening+assays.">Comparative study of membrane potential-sensitive fluorescent probes and their use in ion channel screening assays.</a> J. Biomol. Screen. 8, 533-513 (2003).</li><li>Niswender, C. M., Johnson, K. A., Luo, Q., Avala, J. E., Kim, C., Conn, P. J., Weaver, C. D. <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+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 group III metabotropic glutamate receptors.</a> Mol. Pharm. 73, 1213-1224 (2008).</li><li>Bridal, T. R., Marquilis, M., Wang, X., Donio, M., Sorota, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+Ether-&#225;-go-go+related+gene+screening+assays+based+on+IonWorks+Quattro+and+thallium+flux.">Comparison of human Ether-&#225;-go-go related gene screening assays based on IonWorks Quattro and thallium flux.</a> Assay Drug Dev. Technol. 8, 755-765 (2010).</li></ol>

Mens membran potentiale-følsomme fluorescerende farvestoffer er blevet brugt til at identificere stoffer, som modulerer ionkanaler 9,10, dette er den første rapport af deres ansøgning om neuronal GIRK kanal drug discovery. Den GIRK kanal fluorescenstest præsenteret her tilvejebringer en hurtig, pålidelig og real-time fremgangsmåde til screening af ligand-gatede K +-kanaler. Assayet kan modificeres til anvendelse med en lang række celler, herunder immortaliserede cellelinjer (HEK293, CHO, etc...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> DMEM </td><td> CellGro </td><td> 10-013 </td></tr><tr><td> Hesteserum </td><td> Invitrogen </td><td> 16050-114 </td></tr><tr><td> 96-brøndsplader </td><td> Corning </td><td> 3603 </td></tr><tr><td> Poly-L-lysin </td><td> Sigma-Aldrich </td><td> P4707 </td></tr><tr><td> Somatostatin </td><td> Sigma-Aldrich </td><td> S9129 </td></tr><tr><td> Carbachol </td><td> Sigma-Aldrich </td><td> C4382 </td></tr><tr><td> HLB 021-152 </td><td> Hjælp af Anaspec </td><td> 89300 </td></tr><tr><td> Versette automatiseret væskehåndteringsudstyr </td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> Synergy2 fluorescerende pladelæser </td><td> Biotek </td><td></td></tr><tr><td> Gen5 analyse software </td><td> Biotek </td><td></td></tr></tbody></table> Tabel 1. Tabel af specifikke reagenser og udstyr.

a fluorescent screening assay for identifying modulators of girk channels

G-protein-medierede aktiv ensretter K + (GIRK) kanaler fungerer som cellulære mediatorer af en lang række hormoner og neurotransmittere og udtrykkes i hjerne, hjerte, skeletmuskulatur og endokrine væv 1,2. GIRK kanaler bliver aktiveret, efter at bindingen af ​​ligander (neurotransmittere, hormoner, lægemidler, etc.) til deres plasma membranbundet, G-proteinkoblede receptorer (GPCR&#39;er). Denne binding forårsager stimulering af G-proteiner (G I og G o), som efterfølgende binde til og aktivere GIRK kanal. Efter åbning af GIRK kanalen tillader bevægelse af K + ud af cellen forårsager hvilende membranpotentiale at blive mere negativ. Som følge heraf aftager GIRK kanalaktivering i neuroner spontan virkningspotentialet dannelse og inhiberer frigivelsen af ​​excitatoriske neurotransmittere. I hjertet, hæmmer aktivering af GIRK kanalen pacemakeren aktivitet og dermed bremse puls. <pklasse = &quot;jove_content&quot;&gt; GIRK kanaler repræsenterer nye mål for udvikling af nye terapeutiske midler til behandling af neuropatisk smerte, stofafhængighed, hjertearytmi og andre lidelser 3. Imidlertid farmakologien af ​​disse kanaler er stort set uudforsket. Selv om en række lægemidler, herunder anti-arrhythmiske midler, antipsykotiske lægemidler og antidepressiva blokere GIRK kanal denne inhibering er ikke selektiv og forekommer ved relativt høje lægemiddelkoncentrationer 3. Her beskriver vi en real-time screeningsassay til identifikation af nye modulatorer af GIRK kanaler. I dette assay neuronale AtT20 celler, der udtrykker GIRK kanaler, fyldt med membranpotentialet-sensitive fluorescerende farvestoffer, såsom bis-(1,3-dibutylbarbituric syre) trimethine oxonol [DiBAC 4 (3)] eller HLB 021-152 (figur 1 ). Farvestofmolekylerne bliver stærkt fluorescerende følgende optagelse i cellerne (figur 1). Behandlingaf cellerne med GPCR-ligander stimulerer GIRK kanaler åbnes. Den resulterende K +-efflux ud af cellen får membranen kan blive mere negativ og det fluorescerende signal at falde (figur 1). Således kan lægemidler, som modulerer K +-efflux gennem GIRK kanalen kan analyseres under anvendelse af en fluorescerende pladelæser. I modsætning til andre ion-kanal screening assays, såsom Atomabsorptionsspektrometri 4 eller radiotracer analyse 5, giver GIRK kanalen fluorescenstest en hurtig, real-time og billig screening procedure.

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

En real-tid screening procedure til identifikation af lægemidler, der interagerer med G-protein-gated indad ensretter K<sup&gt; +</sup&gt; (GIRK) kanaler er beskrevet. Assayet anvender membranpotentiale-følsomme fluorescerende farvestoffer til måling GIRK kanalaktivitet. Denne teknik kan tilpasses til anvendelse på en række cellelinier.

En fluorescerende screeningsassay til identifikation af modulatorer af GIRK kanaler

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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