Name: determination of the relative cell surface and total expression of recombinant ion channels using flow cytometry
Uploaded: 2016-09-28T15:00:00
Description: Watch this Scientific Journal Video about Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry at JoVE.com

We thank Mr. Serge S&#233;n&#233;chal and Dr. Jacques Thibodeau for sharing their expertise and granting us access to their flow cytometry and cell sorting platform. This work was completed with the operating grant 130256 from the Canadian Institutes of Health Research, a grant-in-aid from the Canadian Heart and Stroke Foundation, and support from the "Fondation de l'Institut de Cardiologie de Montr&#233;al" to L.P.

1. Dobbelt Tagged DNA konstruktioner <ol><li> Indsæt HA-epitopen (YPYDVPDYA) i det ekstracellulære linker med Ca V α2δ1 mellem D676 og R677 ved site directed mutagenese (figur 3B) 20. Brug Forward primer gccggattatgcgGGAAAACTCCAAACAACC og Reverse primer acatcatacggataTCAATAAATTCATTGAAATTTAAAAGAAATTC. </li><li> Subklone cDNA-sekvensen af den mærkede HA Ca V α2δ1 i den mammale pmCherry-N1 ekspressionsvektor designet til at udtrykke proteinet fusioneret til N-termi...

<ol>
	<li>Delisle, B. P., Anson, B. D., Rajamani, S., January, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+Cardiac+Arrhythmias:+Ion+Channel+Protein+Trafficking.">Biology of Cardiac Arrhythmias: Ion Channel Protein Trafficking.</a> Circ. Res. 94, 1418-1428 (2004).</li><li>Birault, V., Solari, R., Hanrahan, J., Thomas, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correctors+of+the+basic+trafficking+defect+of+the+mutant+F508del-CFTR+that+causes+cystic+fibrosis.">Correctors of the basic trafficking defect of the mutant F508del-CFTR that causes cystic fibrosis.</a> Curr Opin Chem Biol. 17, 353-360 (2013).</li><li>Balijepalli, S. Y., Anderson, C. L., Lin, E. C., January, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+Mutated+Cardiac+Ion+Channels+in+Inherited+Arrhythmia+Syndromes.">Rescue of Mutated Cardiac Ion Channels in Inherited Arrhythmia Syndromes.</a> J. Cardiovas Pharm. 56, 113-122 (2010).</li><li>Gargus, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+Monogenic+Channelopathies+and+Their+Implications+for+Complex+Polygenic+Disease.">Unraveling Monogenic Channelopathies and Their Implications for Complex Polygenic Disease.</a> Am. J. Hum. Genet. 72, 785-803 (2003).</li><li>Abriel, H., Zaklyazminskaya, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+channelopathies:+Genetic+and+molecular+mechanisms.">Cardiac channelopathies: Genetic and molecular mechanisms.</a> Gene. 517, 1-11 (2013).</li><li>Behr, E. 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=Sudden+arrhythmic+death+syndrome:+familial+evaluation+identifies+inheritable+heart+disease+in+the+majority+of+families.">Sudden arrhythmic death syndrome: familial evaluation identifies inheritable heart disease in the majority of families.</a> Eur Heart J. 29, 1670-1680 (2008).</li><li>Catterall, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+regulation+of+voltage-gated+Ca2++channels.">Structure and regulation of voltage-gated Ca2+ channels.</a> Annu. Rev. Cell Dev.Biol. 16, 521-555 (2000).</li><li>Peterson, B. Z., DeMaria, C. D., Adelman, J. P., Yue, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+is+the+Ca2++sensor+for+Ca2++-dependent+inactivation+of+L-+type+calcium+channels.">Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L- type calcium channels.</a> Neuron. 22, 549-558 (1999).</li><li>Dolphin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channel+diversity:+multiple+roles+of+calcium+channel+subunits.">Calcium channel diversity: multiple roles of calcium channel subunits.</a> Curr.Opin.Neurobiol. 19, 237-244 (2009).</li><li>Dai, S., Hall, D. D., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+assemblies+and+localized+regulation+of+voltage-gated+ion+channels.">Supramolecular assemblies and localized regulation of voltage-gated ion channels.</a> Physiol Rev. 89, 411-452 (2009).</li><li>Gao, T., 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=Identification+and+subcellular+localization+of+the+subunits+of+L-type+calcium+channels+and+adenylyl+cyclase+in+cardiac+myocytes.">Identification and subcellular localization of the subunits of L-type calcium channels and adenylyl cyclase in cardiac myocytes.</a> J. Biol. Chem. 272, 19401-19407 (1997).</li><li>Carl, S. L., 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=Immunolocalization+of+sarcolemmal+dihydropyridine+receptor+and+sarcoplasmic+reticular+triadin+and+ryanodine+receptor+in+rabbit+ventricle+and+atrium.">Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium.</a> J. Cell Biol. 129, 673-682 (1995).</li><li>Abriel, H., Rougier, J. S., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+Channel+Macromolecular+Complexes+in+Cardiomyocytes:+Roles+in+Sudden+Cardiac+Death.">Ion Channel Macromolecular Complexes in Cardiomyocytes: Roles in Sudden Cardiac Death.</a> Circ. Res. 116, 1971-1988 (2015).</li><li>Bourdin, B., 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=Molecular+Determinants+of+the+Cavb-induced+Plasma+Membrane+Targeting+of+the+Cav1.2+Channel.">Molecular Determinants of the Cavb-induced Plasma Membrane Targeting of the Cav1.2 Channel.</a> J. Biol. Chem. 285, 22853-22863 (2010).</li><li>Raybaud, A., 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=The+Role+of+the+GX9GX3G+Motif+in+the+Gating+of+High+Voltage-activated+Calcium+Channels.">The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Calcium Channels.</a> J. Biol. Chem. 281, 39424-39436 (2006).</li><li>Burashnikov, E., 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=Mutations+in+the+cardiac+L-type+calcium+channel+associated+with+inherited+J-wave+syndromes+and+sudden+cardiac+death.">Mutations in the cardiac L-type calcium channel associated with inherited J-wave syndromes and sudden cardiac death.</a> Heart Rhythm. 7, 1872-1882 (2010).</li><li>Hennessey, J. A., 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+CACNA1C+Variant+Associated+with+Reduced+Voltage-Dependent+Inactivation,+Increased+Cav1.2+Channel+Window+Current,+and+Arrhythmogenesis.">A CACNA1C Variant Associated with Reduced Voltage-Dependent Inactivation, Increased Cav1.2 Channel Window Current, and Arrhythmogenesis.</a> PLoS ONE. 9, e106982 (2014).</li><li>Adan, A., Alizada, G., Kiraz, Y., Baran, Y., Nalbant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+basic+principles+and+applications.">Flow cytometry: basic principles and applications.</a> Crit Rev Biotechnol. , 1-14 (2016).</li><li>Graham, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Coulter+Principle:+Foundation+of+an+Industry.">The Coulter Principle: Foundation of an Industry.</a> J. Lab. Autom. 8, 72-81 (2003).</li><li>Baumgarth, N., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J. Immunol. Methods. 243, 77-97 (2000).</li><li>Rothe, G., Sack, U., Tarnok, A., Rothe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cellular Diagnostics. Basics, Methods and Clinical Applications of Flow Cytometry. , 53-88 (2009).</li><li>Bourdin, B., 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=Functional+Characterization+of+Cavalpha2delta+Mutations+Associated+with+Sudden+Cardiac+Death.">Functional Characterization of Cavalpha2delta Mutations Associated with Sudden Cardiac Death.</a> J. Biol. Chem. 290, 2854-2869 (2015).</li><li>Tetreault, M. P., 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=Identification+of+glycosylation+sites+essential+for+surface+expression+of+the+Cavalpha2delta1+subunit+and+modulation+of+the+cardiac+Cav1.2+channel+activity.">Identification of glycosylation sites essential for surface expression of the Cavalpha2delta1 subunit and modulation of the cardiac Cav1.2 channel activity.</a> J. Biol. Chem. 291, 4826-4843 (2016).</li><li>Senatore, A., Boone, A. N., Spafford, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Transfection+Strategy+for+Expression+and+Electrophysiological+Recording+of+Recombinant+Voltage-Gated+Ion+Channels+in+HEK-293T+Cells.">Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells.</a> J Vis Exp. (47), (2011).</li><li>Herzenberg, L. A., Tung, J., Moore, W. A., Herzenberg, L. A., Parks, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+flow+cytometry+data:+a+guide+for+the+perplexed.">Interpreting flow cytometry data: a guide for the perplexed.</a> Nat.Immunol. 7, 681-685 (2006).</li><li>Shakeri, B., Bourdin, B., Demers-Giroux, P. O., Sauve, R., Parent, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quartet+of+Leucine+residues+in+the+Guanylate+Kinase+domain+of+Cavbeta+determines+the+plasma+membrane+density+of+the+Cav2.3+channel.">A quartet of Leucine residues in the Guanylate Kinase domain of Cavbeta determines the plasma membrane density of the Cav2.3 channel.</a> J Biol Chem. 287, 32835-32847 (2012).</li><li>Morton, R. A., Baptista-Hon, D. T., Hales, T. G., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonist-+and+antagonist-induced+up-regulation+of+surface+5-HT3A+receptors.">Agonist- and antagonist-induced up-regulation of surface 5-HT3A receptors.</a> Br. J. Pharmacol. 172, 4066-4077 (2015).</li><li>Hoffmann, C., 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=Fluorescent+labeling+of+tetracysteine-tagged+proteins+in+intact+cells.">Fluorescent labeling of tetracysteine-tagged proteins in intact cells.</a> Nat. Protocols. 5, 1666-1677 (2010).</li><li>Cockcroft, C. J., Gamper, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Channels: Methods and Protocols. , 233-241 (2013).</li><li>Gonzalez-Gutierrez, G., Miranda-Laferte, E., Neely, A., Hidalgo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Src+Homology+3+Domain+of+the+beta-Subunit+of+Voltage-gated+Calcium+Channels+Promotes+Endocytosis+via+Dynamin+Interaction.">The Src Homology 3 Domain of the beta-Subunit of Voltage-gated Calcium Channels Promotes Endocytosis via Dynamin Interaction.</a> J. Biol.Chem. 282, 2156-2162 (2007).</li><li>Galizzi, J. P., Borsotto, M., Barhanin, J., Fosset, M., Lazdunski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+photoaffinity+labeling+of+receptor+sites+for+the+Calcium+channel+inhibitors+d-cis-diltiazem,+(+/-)-bepridil,+desmethoxyverapamil,+and+(+)-PN+200-110+in+skeletal+muscle+transverse+tubule+membranes.">Characterization and photoaffinity labeling of receptor sites for the Calcium channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes.</a> J. Biol.Chem. 261, 1393-1397 (1986).</li><li>Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+voltage+sensor+in+voltage-dependent+ion+channels.">The voltage sensor in voltage-dependent ion channels.</a> Physiol.Rev. 80, 555-592 (2000).</li><li>Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+variance+of+sodium+current+fluctuations+at+the+node+of+Ranvier.">The variance of sodium current fluctuations at the node of Ranvier.</a> J Physiol. 307, 97-129 (1980).</li><li>Bailey, M. A., Grabe, M., Devor, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+PCMBS-dependent+modification+of+KCa3.1+channel+gating.">Characterization of the PCMBS-dependent modification of KCa3.1 channel gating.</a> J. Gen. Physiol. 136, 367-387 (2010).</li><li>Fletcher, P. A., Scriven, D. R., Schulson, M. N., Moore, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Image+Colocalization+and+Its+Statistical+Significance.">Multi-Image Colocalization and Its Statistical Significance.</a> Biophys. J. 99, 1996-2005 (2010).</li><li>Lizotte, E., Tremblay, A., Allen, B. G., Fiset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+subcellular+protein+fractions+from+mouse+heart.">Isolation and characterization of subcellular protein fractions from mouse heart.</a> Anal. Biochem. 345, 47-54 (2005).</li><li>Mattheyses, A. L., Simon, S. M., Rappoport, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+with+total+internal+reflection+fluorescence+microscopy+for+the+cell+biologist.">Imaging with total internal reflection fluorescence microscopy for the cell biologist.</a> J. Cell Sci. 123, 3621-3628 (2010).</li><li>Yamamura, H., Suzuki, Y., Imaizumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+light+on+ion+channel+imaging+by+total+internal+reflection+fluorescence+(TIRF)+microscopy.">New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.</a> J. Pharmacol. Sci. 128, 1-7 (2015).</li><li>Wible, B. A., 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=HERG-Lite-R:+A+novel+comprehensive+high-throughput+screen+for+drug-induced+hERG+risk.">HERG-Lite-R: A novel comprehensive high-throughput screen for drug-induced hERG risk.</a> J. Pharmacol. Toxicol. Methods. 52, 136-145 (2005).</li><li>Wilde, A. A. M., Brugada, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypical+Manifestations+of+Mutations+in+the+Genes+Encoding+Subunits+of+the+Cardiac+Sodium+Channel.">Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Sodium Channel.</a> Circ. Res. 108, 884-887 (2011).</li><li>Milano, A., 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=Sudden+Cardiac+Arrest+and+Rare+Genetic+Variants+in+the+Community.">Sudden Cardiac Arrest and Rare Genetic Variants in the Community.</a> Circ Cardiovasc Genet. , (2016).</li><li>Schnell, U., Dijk, F., Sjollema, K. A., Giepmans, B. N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolabeling+artifacts+and+the+need+for+live-cell+imaging.">Immunolabeling artifacts and the need for live-cell imaging.</a> Nat. Meth. 9, 152-158 (2012).</li></ol>

Denne flowcytometri-baseret assay blev anvendt med succes til måling af relativ total og celleoverflade- niveauerne af fluorescens-mærkede poredannende og tilhørende underenheder af spændingsafhængige calciumkanaler 14,22,26. Det er bedst anvendes i forbindelse med undersøgelse af virkningen af ​​genetiske mutationer og kræver således, at den iboende fluorescens intensiteten af ​​den fluorescens-mærkede mærket vildtype-konstruktionen være mindst 10 til 100 gange større end for fluorescensi...

Dette dokument indeholder en pålidelig analyse for at rapportere den relative celleoverfladeekspression af membranproteiner såsom ionkanaler udtrykkes i rekombinante celler under anvendelse af eksisterende flowcytometri teknologi. Lonkanaler er poredannende membranproteiner, der er ansvarlige for kontrollen elektriske signaler ved gating strømmen af ​​ioner gennem cellemembranen. De klassificeres ved aktivering mekanisme, natur, og selektivitet ion arter i transit gennem pore, hvor de er lokaliseret. På de cellulære og væv niveauer, de makroskopiske ionstrømme gennem ionkanaler er et produkt af biofysiske (gating og gennemtrængning), biokemisk (fosforylering), og biogenese (syntese, glykosylering, menneskehandel, og nedbrydning) egenskaber 1. Hver af disse processer er unik for hver type af ionkanaler og er optimeret til at opfylde den fysiologiske rolle af ionkanalen. Derfor ændringer i nogen af ​​disse finjusterede processer gennem enarvet eller en genetisk modifikation, der ofte omtales som &quot;channelopathy&quot;, kan være skadelig for cellehomeostase. Det er vigtigt at understrege, at levere den &quot;rigtige&quot; mængde af ionkanaler ved celleoverfladen er kritisk for cellehomeostase. Selv små stigninger (gain-of-funktion) og små fald (tab af funktion) i ion-kanal aktivitet har potentiale til at forårsage en alvorlig patologi gennem et helt liv. Defekter i celleoverflade levering af modne ionkanaler er en vigtig determinant i talrige kanalopatier, såsom cystisk fibrose (CFTR ionkanal) 2 og hjertearytmier af langt QT-syndrom formular (kardiale kalium kanaler) 3. Kanalopatier er forbundet med hjertestop pludselig død 4. Den nuværende verdensomspændende forekomsten af alle hjerte-kanalopatier menes at være mindst 1: 2,000-1: 3.000 pr person 5 og er ansvarlige for omkring halvdelen af pludselig arytmisk hjertedød cases 6. Dysfunktion i hjertets spændingsstyret natrium-, kalium-, calcium- og selektive ionkanaler er kendt for at spille en central rolle i denne proces. L-typen Ca V 1.2 spændingsstyret calciumkanal til at iværksætte et synkroniseret hjertemusklen sammentrækning. Den kardielle L-typen Ca V 1.2 kanal er en multi-subunit proteinkompleks består af de vigtigste poredannende Ca V α1 underenhed og Ca V ß og Ca V α2δ1 hjælpestoffer underenheder 7-12. Bemærk, at den fuldtallige ekstra underenheder er nødvendig for at producere funktionelle Ca V 1.2 kanaler på plasmamembranen og dynamiske samspil mellem disse underenheder er vigtigt at støtte den normale elektriske funktion af hjertet 13. Ca V ß fremmer celleoverfladeekspression af Ca V 1.2 kanaler gennem en ikke-kovalent nanomolær hydrofob interaktion 14. Co-ekspressionen af Ca V α2δ1 subunit with Ca V SS-bundet Ca V α1 stimulerer spidsstrøm ekspression (5 til 10 gange) og fremmer kanal aktivering ved mere negative spændinger. Gain-of-function mutationer af det poredannende underenhed Ca V 1.2 er blevet forbundet med en form for ventrikulære arytmier kaldet langt QT-syndrom 15 henviser en masse punktmutationer i de tre vigtigste underenheder danner L-typen Ca V 1.2 kanals er blevet identificeret hos patienter, der lider af arytmier af den korte QT-syndrom formular 16,17. Ion-kanaler er membranproteiner, der kan undersøges ud fra et biokemisk synspunkt (protein kemi) eller ved hjælp af elektrofysiologiske værktøjer (strøm-genererende maskiner) og ofte ved hjælp af disse komplementære metoder. Elektrofysiologi, især hel-celle patch-fastspænding, er en egnet fremgangsmåde til at belyse funktionen af ionkanaler 15, men kan ikke løse modifikationer i protein- handel fra ændringer i deres biofysiskeegenskaber. Proteinkemi har imidlertid ofte begrænset anvendelse på grund af den relativt lave ekspression af store membranproteiner i forhold til mindre opløselige proteiner. Robuste high-throughput metoder ved hjælp af fluorescens udlæsning skal udvikles for at specifikt defekter i protein biogenese forårsager ændringer i celleoverfladeekspression af ionkanaler. Flowcytometri er en biofysisk teknologi ansat i celletælling, sortering, biomarkør opdagelse, og protein engineering 18. Når en prøve opløsning af levende celler eller partikler injiceres i et flowcytometer cellerne bestilt i en enkelt strøm, der kan probes ved maskinens detektionssystem (figur 1). Den første flowcytometer instrument produceret i 1956 19 detekteres kun en parameter, men moderne flowcytometre har flere lasere og fluorescensdetektorer der tillader detektion af mere end 30 fluorescerende parametre 20,21.Filtre og spejle (emission optik) lede lysspredning eller fluorescerende lys af celler til et elektronisk netværk (fotodiode og detektorer), der konverterer lyset i forhold til dets intensitet. Digitale data analyseres ved anvendelse af specialiseret software og den primære output vises som et punktdiagram 21. <img alt="figur 1" src="/files/ftp_upload/54732/54732fig1.jpg" /> Figur 1:. Biofysiske principper for flowcytometri sortering Enkelte celler skubbes gennem en dyse under højt tryk i en strøm af sheath fluid, som bevæger dem på en eller flere laser forespørgselssignaler point. Lysstrålen afbøjes af de passerende celler og opsamlet i den fremadgående retning (Forward Scatter, FCS) lys sendes til en fotodiode, der konverterer lyset til et signal proportionalt med størrelsen af ​​cellen. Lyset indsamles også i en 90 ° vinkel i forhold til laser stien og sendes til detektorerne (også kaldet fotomultiplikatorer (PMT)).Dette lys er ført gennem dikroiske spejle, der tillader påvisning af sidespredningen signal (SSC), som afspejler granulering i cellerne, og de fluorescerende emissioner, hvis exciterede fluorochromer er til stede i cellen. Tre detektorer (grøn, gul og rød) er repræsenteret med forskellige bølgelængde båndpasfiltre, muliggør samtidig påvisning af forskellige fluorochromer. De forskellige signaler digitaliseret af en ekstern computer og konverteres til data, der vil blive analyseret for at kvantificere egenskaber cellerne. <a href="http://ecsource.jove.com/files/ftp_upload/54732/54732fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a> Den high-throughput kapacitet flowcytometre blev udnyttet til at kvantificere relative membran ekspression af rekombinant vildtype og handel-deficient spændingsstyret L-typen Ca V 1.2 kanaler og tilknyttede underenheder i levende celler. cDNA-konstruktioner coding for proteinerne blev dobbelt mærkede samtidig at en ekstracellulær ikke-fluorescerende epitop, kan detekteres af en uigennemtrængelig fluorescerende konjugeret antistof og et intracellulært fluorofor, der er konstitutivt fluorescerende. Både den ekstracellulære epitop, indsættes i en ekstracellulær løkke af proteinet, og det intracellulære fluorofor, indsættes efter den C-terminale ende, omregnes med proteinet. I denne serie af forsøg blev Ca V α2δ1 protein manipuleret til at udtrykke en ekstracellulær hemagglutinin (HA) epitop (YPYDVPDYA) detekteres af en uigennemtrængelig FITC (fluorescein isothiocyanat) -konjugeret anti-HA og mCherry som den iboende intracellulære fluorofor. At bestemme den relative celleoverfladeekspression niveau mCherry-Ca V α2δ1 HA-mærket protein blev rekombinante celler, der udtrykker fusionsproteinet høstes efter transfektion, og farvet med FITC-konjugeret monoklonalt muse-anti-HA-epitopmærke Antibody (figur 2). FITC er en organisk fluorescerende forbindelse, der er betydeligt mindre end enzym journalister, og derfor ikke så tilbøjelige til at forstyrre biologisk funktion. mCherry- Ca V α2δ1-HA overudtrykt i Tsa-201cells, producerer en signifikant 3-log stigning i FITC fluorescens og mCherry fluorescens på todimensionale plots 22. Da HA-epitopen ligger i den ekstracellulære del af proteinet, fluorescensintensiteten for FITC opnået i nærvær af intakte celler afspejler den relative indeks for celleoverfladeekspression af HA-mærket protein. Tilgængeligheden af ​​HA-epitopen i konstruktionerne systematisk valideres ved måling af FITC signal efter celle permeabilisering. Denne foranstaltning tjener også til at bekræfte den normaliserede total protein udtryk, da de relative fluorescensintensiteter for FITC estimeret i permeabiliserede celler er kvalitativt sammenlignelig med de relative fluorescens værdier for mCherry målt under permeabiliserede og ikke-permeabiliseres betingelser 22,23. Det er vigtigt at bemærke, at den iboende fluorescensspektret forskydes mod højere værdier efter permeabilisering, men at den eneste værdi, der rapporteres er ændringen i fluorescensintensitet i sammenligning med kontrolgruppen konstruktionen. Relative ændringer i fluorescensintensitet for test konstruktioner estimeres ved brug af ΔMean Fluorescence Intensity (ΔMFI) værdier for hver fluoroforen (mCherry eller FITC). Eksperimenter er designet til at måle fluorescensintensiteten af ​​testen konstruktionen i forhold til fluorescensintensitet kontrol konstrukt udtrykkes under de samme betingelser for at begrænse eksperimentelle variationer i den iboende fluorescens fra fluoroforen-konjugerede antistof. To membranproteiner lykkedes undersøgt ved anvendelse af dette assay: det poredannende underenhed af L-typen spændingsstyret calciumkanal Ca V 1.2 14,22 og i en anden serie afeksperimenter, den ekstracellulære ekstra Ca V α2δ1 subunit 22,23. Den følgende protokol blev anvendt til at bestemme celleoverfladeekspression af Ca V α2δ1 underenhed af hjerte- L-typen Ca V 1.2 kanal under kontrolbetingelser og efter mutationer påvirker den posttranslationelle modifikation af ionkanalen. Under standardiserede forsøgsbetingelser, celleoverfladen fluorescens af FITC øger kvasi-lineært med ekspressionen af cDNA, der koder for mCherry-Ca V α2δ1-HA-proteiner (figur 5 fra henvisning 22). <img alt="Figur 2" src="/files/ftp_upload/54732/54732fig2.jpg" /> Figur 2:. Skematisk fremstilling af total og membran mærkning i flowcytometri forsøgsprotokollen Ordningen skitserer nogle af de vigtigste skridt, der er nødvendige for at kvantificere den relative totale og celleoverfladen ekspression af rekombinante ionkanaler ved flow cytometri. Celler transficeret med dobbelt-mærket konstruktion mCherry-Ca V α2δ1-HA i Tsa-201-celler (1) og farvet før eller efter permeabilisering (2). Multiparameter data er erhvervet i et flowcytometer (3) for multivariat analyse (4). <a href="http://ecsource.jove.com/files/ftp_upload/54732/54732fig2large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a>

<table border="0" cellpadding="0" cellspacing="0" width="763">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td style="width:143px;">New England Biolabs</td>
			<td style="width:123px;">E0554S</td>
			<td style="width:281px;">Can be substitute with QuickChange site-directed mutagenesis Kit (Agilent, #200523).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubes 1,5 mL</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">72-690-001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubes 15 mL&#160;</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">&#160;62-554-002</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable graduated Tranfer Pipets&#160;</td>
			<td style="width:143px;">VWR</td>
			<td style="width:123px;">160001-192&#160;</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100-mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:123px;">430167</td>
			<td style="width:281px;">For standard culture of HEKT cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35-mm culture dish</td>
			<td style="width:143px;">Falcon</td>
			<td style="width:123px;">353001</td>
			<td style="width:281px;">For standard culture of HEKT cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 1 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1251.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 5 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1253.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 10 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1254.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 25ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1285.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dulbecco&#39;s high-glucose medium</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">12100-046</td>
			<td style="width:281px;">Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fetal Bovine Serum, qualified, heat inactivated, US origin</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">16140-071</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">15140-122</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Lipofectamine 2000&#160;</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">11668-019</td>
			<td style="width:281px;">For liposomal transfection. Can be substituted with calcium phosphate transfection.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">31985-070</td>
			<td style="width:281px;">Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Trypsin-EDTA (1X) 0.05%, phenol red</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">25300-062</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL microtubes</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">72.690.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Phosphate Buffered Saline 1X</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">BP661-10</td>
			<td style="width:281px;">Can be &#34;home-made&#34;.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Anti-HA FITC conjugated antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:123px;">H7411</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">IgG1&#8722;FITC Isotype Control antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:123px;">F6397</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="20">
			<td height="61" rowspan="2" style="height:61px;width:216px;">BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:123px;">554714</td>
			<td style="width:281px;">Fixation/Permeabilization. Permeabilization/Wash solution, store at 4 &#176;C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:143px;"></td>
			<td style="width:123px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">49105161</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">15250061</td>
			<td style="width:281px;">To access cell viability.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Refrigerated Microcentrifuge, 5430R</td>
			<td style="width:143px;">Eppendorf&#160;</td>
			<td style="width:123px;">A14H172200</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Forma Steri-Cycle CO2 Incubator</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">370</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laboratory Platform Rocker</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">545034</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water Bath</td>
			<td style="width:143px;">VWR</td>
			<td style="width:123px;">89032-216</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD FACSARIA III&#160;</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:123px;">648282</td>
			<td style="width:281px;">Flow cytometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FlowJo Software v10</td>
			<td style="width:143px;">FlowJo</td>
			<td style="width:123px;">FlowJo v10 Dongle</td>
			<td style="width:281px;">For data analysis.</td>
		</tr>
	</tbody>
</table>

determination of the relative cell surface and total expression of recombinant ion channels using flow cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

I denne artikel beskrives en pålidelig protokol til at kvantificere totale og celleoverfladen af ​​rekombinante ionkanaler udtrykt i Tsa-201cells af en tofarvet flowcytometri assay. Som et eksempel blev den relative celleoverfladen og totalt protein ekspression kvantificeres for Ca V α2δ1subunit. For at udføre den tofarvede flowcytometri assay blev Ca V α2δ1 dobbelt mærkede at udtrykke en ekstracellulær ikke-fluorescerende epitop HA, der kan påvises ved...

Watch this Scientific Journal Video about Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry at JoVE.com

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Nedarvede hjertearytmier er ofte forårsaget af mutationer, som ændrer overfladen tilførsel af ét eller flere ionkanaler. Her, tilpasser vi flowcytometri assays til tilvejebringelse af en kvantificering af den relative totale og celleoverfladeprotein ekspression af rekombinante ionkanaler udtrykkes i Tsa-201 celler.

Bestemmelse af den relative celleoverfladen og Total ekspression af rekombinante Ion Channels anvendelse af flowcytometri

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Research

JoVE Journal

Biology

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Vurdering af GFP Expression og levedygtighed Brug af Tali Billed-Based cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Aviditet baseret ekstracellulær interaktion Screening (AVEXIS) for skalerbar detektion af lav affinitet Ekstracellulære Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flowcytometri-baseret oprensning af<em&gt; S. cerevisiae</em&gt; Zygoter

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...