Een proteoliposome-Based Efflux Assay om Enkelmolecuul Eigenschappen van Cl Bepaal<sup> -</sup> Kanalen en Transporters
Summary
Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.
Abstract
The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl– channels or transporters is described. The assay is based on the measure of the efflux rate of Cl– from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl– exchanger, as a model system. The efflux assay can be utilized to study any Cl– conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.
Introduction
In de laatste twee decennia is de mogelijkheid om overexpressie en te zuiveren membraan transporteiwitten is dramatisch toegenomen: ionkanalen, primaire en secundaire transporteurs worden nu routinematig gezuiverd van heterologe expressie systemen, alsmede natuurlijke bronnen. Nieuwe benaderingen te controleren meningsuiting, te verbeteren en de winning faciliteren en versterken de stabiliteit van deze eiwitten worden constant ontwikkeld 1-5. Deze technologische doorbraken zijn geweest bij het activeren van de explosie van atoom-niveau structurele informatie over membraaneiwitten die op zijn beurt verbeterde ons begrip van de structurele basis van hun functie is. Daarentegen ons vermogen sonderen de functionele eigenschappen van de gezuiverde eiwitten geen verhoging in hetzelfde tempo, zodat in sommige gevallen hoge resolutie structuurinformatie gepaard met kwalitatieve functionele gegevens, waardoor ons vermogen om kwantitatief te testen structuur gebaseerde voorspellingen beperkt. Vandaar dat de development van kwantitatieve en generaliseerbaar functionele assays is een belangrijke stap naar de ontrafeling van de mechanistische onderbouwing van membraaneiwit functie.
Hier beschrijven we een efflux assay die kan worden gebruikt om kwantitatief belangrijkste functionele eigenschappen van gezuiverd en gereconstitueerd Cl – kanalen en transporters. De beginselen van de test kan worden gegeneraliseerd naar diverse transportsystemen, alsmede niet-ion transport eiwitten. Liposomen worden gereconstitueerd met gezuiverde Cl – kanaal / transporters in de aanwezigheid van een grote Cl – helling (figuur 1A, B). Cl – efflux wordt geïnitieerd door de toevoeging van een ionofoor om voor tegenion flux, in ons geval de K + ionofoor valinomycine, dat voldoet aan de Cl spanning shunt – gradiënt en het beginvolume membraanpotentiaal de evenwichtspotentiaal van K + 6,7. Zonder thij ionofoor geen significant netto Cl – efflux optreedt, zoals wordt voorkomen door het genereren van een transmembraanpotentiaal. De data wordt kwantitatief beschreven door twee meetbare parameters (figuur 1C): τ de tijdconstante van Cl – efflux en f0, de fractie van liposomen actief eiwit bevatten. Van τ en f 0 de unitaire Cl – transportsnelheid, kan de fractie van actief eiwit en de molecuulmassa van het actieve complex afgeleid 8. De techniek wordt hier geïllustreerd aan de hand proteoliposomen gereconstitueerd met CLC-EC1, een goed gekarakteriseerde CLC-type H + / Cl – wisselaar van bekende structuur en functie. Deze test wordt gemakkelijk gegeneraliseerd om kanalen of transporters met verschillende ionische selectiviteit of waarvan de activiteit afhankelijk van de aanwezigheid van spanning en / of liganden. Bovendien kan deze bepaling worden vastgesteld of kleine moleculen rechtstreeks de gereconstitueerde eiwit,om de effecten van deze verbindingen en hoe membraansamenstelling of lipidemodificerend reagentia invloed op de functie van de gereconstitueerde kanalen en transporters kwantificeren.
Protocol
Representative Results
Discussion
Transport gemedieerd door gezuiverd anion-selectieve kanalen of transporteurs gereconstitueerd in liposomen – We hebben een gedetailleerd protocol om Cl te meten beschreven. Het voorbeeld gebruikt was de prokaryotische H + / Cl – wisselaar CLC-EC1. Echter, kan de methode gemakkelijk worden aangepast om kanalen afgesloten door liganden 12,13,15, 11,12 spanning of sportieve andere anionogene selectiviteit 15,16 door vervanging van de Ag bestuderen: AgCl elektrode met …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Liposomicator, Avanti Polar Lipids Inc. | Avanti Polar Lipids Inc. | 610200 | |
| IEC Centra CL2 Benchtop | Thermo Scientific | ||
| Orion Research Model 701A Digital pH-mV meter | These can be found on Ebay. | ||
| Non-functional pH probe | Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned. | ||
| DI-710 Data Logger | DATAQ instruments | ||
| WinDAQ acquisition software | DATAQ instruments | ||
| Pierce Disposable Plastic Columns, Gravity-flow, 2ml | Pierce (Thermo Scientific) | 29922 | |
| KIMAX Culture Tubes, Disposable, Borosilicate Glass | Kimble Chase | 73500-13100 | |
| Extruder Set With Holder/Heating Block | Avanti Polar Lipids Inc. | 610000 | |
| Computer |
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