Name: proteomics to identify proteins interacting with p2x2 ligand-gated cation channels
Uploaded: 2009-05-18T23:00:00
Description: Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

SW en TMV worden ondersteund door de NCRR en NHLBI bij de National Institutes of Health. BSK en HS worden ondersteund door de NINDS en NIGMS van de National Institutes of Health.

Experimentele procedures De experimentele procedure (figuur 2) bestaat uit vier onderdelen die hieronder worden beschreven in een stapsgewijze manier. Deel 1: subkloneren en expressie van de C-terminus van P2X2 receptoren. We hebben geuit over de volle lengte C-terminus van P2X2 receptoren in bacteriën naar de hersenen eiwitten waaraan het bindt identificeren. <ol><li> De C-terminus (residuen 353-472) van de P2X2 receptor (figuur 1) ...

<ol>
	<li>Hille, B. <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 of excitable membranes. , (2001).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors+and+ATP+signalling+at+synapses.">Molecular physiology of P2X receptors and ATP signalling at synapses.</a> Nat Rev Neurosci. 2, 165-174 (2001).</li><li>Egan, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+calcium+ions+to+P2X+channel+responses.">Contribution of calcium ions to P2X channel responses.</a> J Neurosci. 24, 3413-3420 (2004).</li><li>Burnashev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+permeability+of+ligand-gated+channels.">Calcium permeability of ligand-gated channels.</a> Cell Calcium. 24, 325-332 (1998).</li><li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131, 1047-1058 (2007).</li><li>Levitan, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+protein+complexes+associated+with+neuronal+ion+channels.">Signaling protein complexes associated with neuronal ion channels.</a> Nat Neurosci. 9, 305-310 (2006).</li><li>Khakh, B. S., North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X+receptors+as+cell-surface+ATP+sensors+in+health+and+disease.">P2X receptors as cell-surface ATP sensors in health and disease.</a> Nature. 442, 527-532 (2006).</li><li>Roger, S., Pelegrin, P., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+P2X7+receptor+currents+and+membrane+blebbing+via+constitutive+and+dynamic+calmodulin+binding.">Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding.</a> J Neurosci. 28, 6393-6401 (2008).</li><li>North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors.">Molecular physiology of P2X receptors.</a> Physiol Rev. 82, 1013-1067 (2002).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+union+of+pharmacology.+XXIV.+Current+status+of+the+nomenclature+and+properties+of+P2X+receptors+and+their+subunits..">International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits..</a> Pharmacol Rev. 53, 107-118 (2001).</li><li>Young, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+shape,+architecture+and+size+of+P2X4+receptors+determined+using+fluorescence+resonance+energy+transfer+and+electron+microscopy.">Molecular shape, architecture and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.</a> J Biol Chem. 283, 26241-26251 (2008).</li><li>Edmondson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+signaling+complexes+include+metabolism-+and+transcription/translation-related+proteins:+complimentary+separation+techniques+with+LC/MS/MS.">Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS.</a> Mol Cell Proteomics. 1, 421-433 (2002).</li><li>Evans, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthosteric+and+allosteric+binding+sites+of+P2X+receptors.">Orthosteric and allosteric binding sites of P2X receptors.</a> Eur Biophys J. 38, 319-327 (2009).</li></ol>

Ion kanalen zijn een belangrijke klasse van integrale membraaneiwitten. Ze bevatten met water gevulde poriën die selectief toestaan ​​dat de beweging van ionen neer hun elektrochemische gradiënten het plasmamembraan. Ionkanalen hek tussen open en de gesloten staten. De gating stap wordt geactiveerd door zenders (bijv. ATP) in geval van P2X ligand gated ionkanalen, of het kan worden gereguleerd door interacties met andere eiwitten. Het laatste decennium is getuige geweest van een toename van ons begrip van hoe P2X ...

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proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

Ligand-gated ionkanalen ten grondslag liggen aan de synaptische communicatie in het zenuwstelsel 1. Bij zoogdieren zijn er drie families van ligand-gated kanalen: de Cys lus, de glutamaat-gated en de P2X receptor kanalen 2. In elk geval binding van de zender leidt tot de opening van een porie waardoor ionen naar beneden stromen hun elektrochemische gradiënten. Veel ligand-gated kanalen zijn ook doorlaatbaar voor calciumionen 3, 4, die stroomafwaarts hebben signalering rollen 5 (bijv. gen regulatie), die de duur van het kanaal opening kan overschrijden. Zo ligand-gated kanalen kunnen signaal over een brede tijdschalen variërend van enkele milliseconden tot enkele dagen. Gezien deze belangrijke rol is het noodzakelijk om te begrijpen hoe ligand-gated ionkanalen zelf worden geregeld door eiwitten, en hoe deze eiwitten kunnen afstemmen signalering. Recente studies tonen aan dat vele, zo niet alle, kanalen kan een deel van eiwit signalisatie complexen 6. In dit artikel leggen we uit hoe je de eiwitten die binden aan de C-terminale aspecten van de P2X2 receptor cytosolische domein te identificeren. P2X receptoren zijn ATP-gated kation kanalen en bestaat uit zeven subeenheden (P2X1-P2X7). P2X receptoren worden op grote schaal tot uitdrukking in de hersenen, waar ze bemiddelen excitatoire synaptische transmissie en presynaptische facilitatie van neurotransmitters 7. P2X receptoren zijn gevonden in prikkelbaar en niet-opgewonden cellen en bemiddelen belangrijke rol bij neuronale signalering, ontstekingen en cardiovasculaire functie 8. P2X2 receptoren zijn overvloedig in het zenuwstelsel 9 en zijn de focus van deze studie. Elke P2X subeenheid wordt gedacht aan twee membraan verspreid over segmenten (TM1 en TM2), gescheiden door een extracellulaire regio 7 en intracellulaire N-en C-termini (afb. 1a) 7 bezitten. P2X subunits 10 (P2X1-P2X7) tonen aan 30-50% sequentiehomologie op het aminozuur level 11. P2X receptoren bevatten slechts drie subeenheden, dat is de eenvoudigste stoichiometrie onder ionotrope receptoren. De P2X2 C-terminus bestaat uit 120 aminozuren (Figuur 1b) en bevat verschillende eiwitten docking consensus sites, ondersteunen de hypothese dat P2X2 receptor kan deel uitmaken van signalering complexen. Hoewel een aantal functies zijn toegeschreven aan de C-terminus van P2X2 receptoren 9 geen enkele studie heeft beschreven de moleculaire partners die paar om de intracellulaire kant van dit eiwit via de volledige lengte C-terminus. In deze methoden paper beschrijven we een proteoom benadering van de proteïnen die interageren met de volledige lengte C-terminus van P2X2 receptoren te identificeren.

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Beschrijven we een eenvoudig protocol om de hersenen eiwitten die zich binden aan de volledige lengte C-terminus van de ATP-gated P2X2 receptoren te identificeren. De uitbreiding en systematische toepassing van deze aanpak op alle P2X receptoren zal naar verwachting leiden tot een beter begrip van P2X receptor signalering.

Proteomics om eiwitten Interactie met P2X2 ligand-gated kationenkanalen identificeren

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

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