Name: using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking
Uploaded: 2014-03-28T15:45:00
Description: Watch this Scientific Journal Video about Using an ÃŽÂ±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Der blev leveret af startup-midler fra Farmakologi og Chemical Biology Department på University of Pittsburgh School of Medicine. Anerkendelse af Jacob lab medlemmer, der har bidraget til video indsendelse: Nicholas Graff.

Alle protokoller, der er beskrevet nedenfor, er i overensstemmelse med IACUC og IRB Institutional Review bestyrelser University of Pittsburgh School of Medicine. 1.. Udarbejdelse af Hippocampus neuronkulturer i vævskultur Hood Bemærk: Brug steril teknik og reagenser hele protokol 1. <ol><li> Forbered poly-D-lysin (0,1 mg / ml i H2O) coatede dækglas som et substrat for den neuronale kultur. <ol><li> Placer 4-5 runde dækglas inde hver 3,5 cm vævskulturskål. </li><li>...

<ol>
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BBS baserede faste og levende teknikker beskrevet her kan anvendes til at spore receptor eller anden plasmamembranprotein handel med cellelinier, neuroner og andre primære celler. Denne fremgangsmåde er med succes blevet brugt til at studere membran indsættelse og fjernelse af ligand-gatede ionkanaler og GPCR og vurdere ændringer i handel på grund af tilstedeværelsen af ​​receptoragonister og modulatorer. Vigtige aspekter omfatte passende lokalisering af tag til et ekstracellulært sted og udføre kontroller f...

Anvendelsen af fluorescens koblet α-bungarotoxin for at studere receptor lokalisering og dynamik var banebrydende ved undersøgelser af nikotinacetylcholinreceptoren 13-15 toksinet endogene mål. Efterfølgende har inkorporering af den minimale Bgt bindende peptid (BBS) blevet anvendt til at undersøge handel med både stimulerende og hæmmende ligandstyrede ionkanaler og G proteinkoblede receptorer 2,10,16-21. Dette BBS-baserede teknik giver fordele til andre tilgange, der anvendes til undersøgelser for menneskehandel, såsom overflade biotinyleringsbetingelserne metoder, antistof-mærkning af levende celler med antistoffer mod ekstracellulære epitoper, og fluorescens bedring efter fotoblegning (FRAP). Under celleoverfladen biotinyleringsbetingelserne frie aminer er kovalent modificeret med potentiale til at påvirke cellulær aktivitet. Antistof baserede undersøgelser er ofte blevet hæmmet af overfladeantigen clustering eller capping, som kan ændre begivenheder smuglerruter. På grund af blegning skridt for FRAP studies er et vigtigt indsatsområde at beskadige den underliggende cellestruktur. En yderligere fordel er, at BBS mærkede konstruktioner kan også anvendes til biokemiske metoder med biotin-koblede bungarotoxin at vurdere receptor handel. Denne teknik er let anvendelse på cellelinier og primære celler. Til brug i celler, der udtrykker nicotin-acetylcholin (nAChR)-receptorer, skal nAChR antagonist tubocurarin bruges i hele protokollen som angivet. Udførelse af en simpel overflade Bgt etikettering (svarende til T = 0 tidspunkt for endocytose protokol) på utransficerede celler i fravær af tubocurarin vil fremlægge dokumentation for endogent nAChR. En vigtig overvejelse ved anvendelse af denne teknik er passende indsættelse af BBS, så det er til stede i en ekstracellulær placering, når proteinet af interesse er leveret til plasmamembranen. For eksempel N-terminale domæner af GABAAR underenheder bosat i vesikulære lumen under trafikskehandel og bliver ekstracellulære efter receptor indsættelse i plasmamembranen, så specifik mærkning af celleoverfladereceptorer og vurdering af deres fjernelse fra celleoverfladen ved endocytiske begivenheder. Vi har tidligere vist, at tilsætningen af ​​GFP, myc eller BBS epitoper til dette domæne af GABAAR underenheder er funktionelt tavse. Standard kontroller bør udføres for at sikre, at den mærkede protein er udtrykt på samme niveau til et ukodet konstruktion, at det korrekt lokaliseret, og at det ikke påvirker receptor funktion. Denne karakteristik af transfekterede konstruktioner vil også støtte i fejlfinding overekspression bekymringer.

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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
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			<td height="20" style="height:20px;width:227px;">anti GFP</td>
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using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

Det er i stigende grad klart, at neurotransmitterreceptorer, herunder ionotropic GABA A receptorer (GABAAR), udviser meget dynamisk handel og celleoverfladen mobilitet 1-7. For at undersøge receptor celleoverfladen lokalisering og endocytose, den her beskrevne teknik kombinerer brugen af ​​fluorescerende α-bungarotoxin med celler, der udtrykker konstruktioner indeholdende en α-bungarotoxin (Bgt) bindingssted (BBS). BBS (WRYYESSLEPYPD) er baseret på α-underenheden af musklen nikotinacetylcholinreceptoren, som binder Bgt med høj affinitet 8,9. Inkorporering af BBS hjemmeside giver overflade lokalisering og måling af receptor indsættelse eller fjernelse med anvendelse af eksogene fluorescerende Bgt, som tidligere beskrevet i sporing af GABAA og metabotropisk GABAB receptorer 2,10. Ud over BBS site, vi indsat en pH-følsom GFP (pHGFP 11) mellem aminosyrerne 4 og 5 af det modne GABAAR underenheden af standard-molecular biologi og PCR kloningsstrategier (se figur 1) 12. BBS er 3 &#39;af pH-følsomme GFP reporter, adskilt af en 13-aminosyren alanin / prolin-linker. For trafficking undersøgelser er beskrevet i denne publikation, der er baseret på faste prøver det pHGFP fungerer som en reporter af alt mærkede GABAAR subunit protein niveauer, så normalisering af Bgt mærkede receptor population til den samlede receptor befolkning. Dette minimerer celle til celle Bgt farvning signal variation som følge af højere eller lavere baseline udtryk for de mærkede GABAAR underenheder. Desuden pHGFP tag gør det let at identificere konstruere udtrykker celler til levende eller faste billeddannelse eksperimenter.

Karakterisering af en BBS mærkede konstruktion omfatter vigtige kontroller såsom bestemmelse af, om det udtrykte protein samles korrekt (især med receptorer er sammensat af flere underenheder), trafikker til celleoverfladen og lokaliseres hensigtsmæssigt. Hæmmende synapser er sammensat af GABA A receptor overflade klynger, colocalize med den hæmmende stillads protein gephyrin og apposed til præsynaptiske hæmmende terminaler, identificeret af vesikulær hæmmende aminosyretransporter der indlæser GABA og glycin ...

Watch this Scientific Journal Video about Using an ÃŽÂ±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an ÃƒÅ½Ã‚Â±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Ved hjælp af en α-Bungarotoxin-bindingssted Tag til Study GABAA receptor membranlokalisering og handel

Her har vi demonstrere brugen af ​​fluorescerende Alexa farvestof koblet til α-bungarotoxin at måle GABAA receptor overflade lokalisering og endocytose i hippocampus neuroner. Gennem brug af konstruktionerne, der bærer en kort ekstracellulære tag, der binder α-bungarotoxin, kan opnås analyse af plasmamembranprotein endocytiske handel.

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

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Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

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