En proteoliposom-Based udstrømning Assay til bestemmelse Single-molekyle egenskaber Cl<sup> -</sup> Kanaler og transportører
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
I de sidste to årtier evnen til at overudtrykke og rense membrantransportproteiner steget dramatisk: ionkanaler, primære og sekundære transportører nu rutinemæssigt oprenset fra heterolog ekspression systemer samt naturlige kilder. Nye metoder til at overvåge udtryk, forbedre og lette udvindingen og øge stabiliteten af disse proteiner er konstant udvikles 1-5. Disse teknologiske gennembrud har været medvirkende til at udløse eksplosionen af atomart niveau strukturel information om membranproteiner, som til gengæld forbedret vores forståelse af de strukturelle grundlag for deres funktion. I modsætning til vores evne sonde de funktionelle egenskaber af de oprensede proteiner ikke øges ved den samme hastighed, så at i nogle tilfælde høj opløsning strukturel information er ledsaget af kvalitative funktionelle data, hvilket begrænser vores evne til kvantitativt teststrukturen-baserede forudsigelser. Derfor er UDVIKLINGSt af kvantitative og generalisere funktionelle assays er et vigtigt skridt i retning af opklaringen af de mekanistiske fundament for membranprotein funktion.
Her beskriver vi en efflux assay, der kan anvendes til kvantitativt at bestemme vigtige funktionelle egenskaber af oprenset og rekonstituerede Cl – kanaler og transportører. De principper analysen kan generaliseres til en række forskellige transportsystemer samt til ikke ion-transport proteiner. Liposomer rekonstitueres med oprensede CL – kanal / transportører i nærvær af et stort Cl – gradient (figur 1A, B). Cl – efflux initieres ved tilsætning af en ionofor for at tillade counter-ion flux, i vores tilfælde K + ionofor valinomycin, som shunt spændingen oprettet ved Cl – gradient og indstille den indledende membranpotentialet til ligevægt potentiale K + 6,7. Uden than ionofor ingen signifikant net Cl – udstrømning sker, som det er forhindret ved genereringen af et transmembrant potentiale. Dataene kvantitativt beskrevet af to målbare parametre (figur 1C): τ, den tidskonstant Cl – efflux og f 0, den del af liposomer, der ikke indeholder et aktivt protein. Fra τ og f 0 enhedskarakter Cl – transport rate, kan den del af aktive proteiner og den molekylære masse af det aktive kompleks afledes 8. Teknikken er illustreret her ved hjælp af proteoliposomer rekonstitueret med CLC-EC1, en velkarakteriseret CLC-typen H + / Cl – varmeveksler af kendt struktur og funktion. Denne analyse er let generaliseres til kanaler eller transportører med forskellige ioniske selektivitet eller hvis aktiviteter afhænger af tilstedeværelsen af spænding og / eller ligander. Desuden kan dette assay anvendes til at bestemme, om små molekyler direkte påvirke rekonstituerede protein,at kvantificere virkningerne af disse forbindelser og hvordan membransammensætning eller lipid-modificerende reagenser påvirker funktionen af de rekonstituerede kanaler og transportører.
Protocol
Representative Results
Discussion
Vi har beskrevet en detaljeret protokol til at måle Cl – transport medieret af oprensede anion-selektive kanaler eller transportører rekonstitueret i liposomer. Eksemplet anvendte var den prokaryote H + / Cl – veksleren CLC-EC1. Imidlertid kan metoden let tilpasses til at studere kanaler gated af ligander 12,13,15, spænding 11,12 eller sportslige forskellige anioniske selektivitet 15,16 ved at erstatte Ag: AgCl elektrode med en egnet for ionen under ov…
Disclosures
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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