Name: a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters
Uploaded: 2015-04-20T13:00:00.000+00:00
Duration: 7 min 47 s
Description: Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1. Lipid Preparasyonu <ol><li> Saydam bir cam tüp içine istenilen miktarda lipit bölmeyin. Kullanım E. E. coli kutuplu lipit özü, ama en lipit bileşimler kullanılabilir. Lipidler toz biçiminde ise, 20 mg / ml &#39;lik bir konsantrasyona kadar kloroform bunları yeniden süspanse edin. Bütün solvent buharlaşana kadar N2 gazı altında oda sıcaklığında lipitler kurutun. </li><li> Pentan lipitleri tekrar ve kloroform kalan izleri kaldırmak için yeniden gaz N 2 akışı kurutun. Kuru bütün solvent çekinceye kadar. Kurutma sırasında, lipid altındaki bir biçim verme yoğun kütle yerine borunun alt üçte birini örtmekte olan eşbiçimli bir film dağıtılır ve böylece tüp hafifçe döndürün. </li><li> Sulandırma tamponu içeren bir deterjan ekleme (300 mM KCI, 50 mM sitrik asit, 25 mM K 2 HPO 4, pH 4.5, 35 mM CHAPS) lipidler eritin. Protein CHAPS&#39;tan kötü hoşgörülü ise bu adım için diğer hafif deterjanlar kullanın. </li><li> ResuspenBir su banyosu sonikatör kullanarak netlik lipidler d. Kısa (30 sn-1 dk) ile kısa (30 sn-1 dk) bakliyat maksimum güçte su banyosunda sonikatör merkezinde tüp daldırın duraklar. Çözelti berrak hale gelmelidir. Not: açıklık son derece elde kullanılan spesifik yağ bileşimine bağlıdır. Da nominal olarak denk lipidler arasında bu adımda bazı partiden partiye değişkenlik, aynı zamanda mümkündür. Kalıntı bulanıklık santrifüjleme ile çıkarılabilir süspansiyonda büyük parçacıklar tarafından ışık saçılımı kaynaklanmaktadır. </li><li> 20-60 dakika boyunca oda sıcaklığında yeniden süspansiyon haline lipitler inkübe edin. </li></ol> 2. Proteoliposome Formasyonu Not: birkaç strateji lipozomlara deterjan çözülmüş protein eklemek için kullanılabilir. CLC-EC1 için diyaliz iyi çalışır ve bu nedenle tercih 6,9,10 bir yöntemdir. <ol><li> Saflaştırılmış protein ekleyin istenen protein-to-lipid ug o ifade (P / L) oranı (<html> F proteinine mg / lipid). P / L oranının seçimi deney amacına bağlıdır. <ol><li> Amaç, ilgi konusu proteinin aktivitesini değerlendirmek yüksek P tekrar oluşturmak için, her lipozom proteininin birden fazla kopyasını içerdiğinden / L ile, sinyali en üst düzeye çıkarmak için. Her bir kesecik ortalama 1 protein ihtiva edecek şekilde aktivitesi ve / veya proteinin üniter nakil oranını ölçmek için, düşük P / L&#39;nin de bir Poisson seyreltme rejiminde sulandırın. Her rejimi kullanmak için ne zaman bir daha derinlemesine analiz için Adım 7 ve Tartışma bakın. </li></ol></li><li> Farklı tedavi rejimleri için uygun P / L değerlerini belirlemek için, protein konsantrasyonu 8,11-13 bir fonksiyonu olarak aktivitesi tam bir titrasyon yapmak. Bununla birlikte, aktif biriminin moleküler ağırlığı (MW kDa olarak belirtilmiştir) için kullanılan herhangi bir protein için, aşağıdaki P / L değerleri ilk guesstimates olarak kullanılabilir bilinmektedir: pg &quot;/&gt; <img alt="Denklem 2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li> Önceden ıslatılmış diyaliz tüp veya bir kaset protein ve lipit karışımı yükleyin. 500-1,000x lipit yeniden oluşturma tampon hacmi ihtiva eden bir beher içinde diyaliz cihazı yerleştirin (yani, yağ yeniden süspanse edilmesi için 1 ml 1 L tamponu) hafif karıştırma altında. <ol><li> Değişim tampon aralıkları&gt; 3 saatte 3-4 kez. Her çözelti, değişiminde, kaset / boru ve damıtılmış su ile beher yıkayın ve taze tampon maddesi ile yeniden oluşturma beher doldurun. Çözelti değişikliği için 1-2 O / N aralıklarını ekleyin. çözüm değişiklikleri tam sayısı ve süresi kullanılan tam lipid ve deterjanlar bağlıdır. </li></ol></li><li> Son adım tamamlandıktan sonra, diyaliz tekneden lipozomlar kaldırmak tüpler onları bölmeyin ve flaş -80 ° C&#39;de sıvı N 2 veya dondurularak onları dondurmak. </li></ol> 3. Kayıt Ayarı &lt;p class = &quot;jove_content&quot;&gt; NOT: Kayıt set-up (Şekil 2A) iki odadan (düz tabanlı silindir, ~ 3-4 ml hacimli), bir Cl oluşur - elektrot, bir analog bir pH metre (aşağıya bakınız) dijital elektrik çıkışı, bir sayısallaştırıcı ve uygun bir satın alma yazılımı ile bilgisayar veya. <ol><li> PH metre elektrodu, bilgisayara bağlı olan sayısallaştırıcıya pHmetrenin çıkışını - Cl bağlayın. Bir odasında referans elektrot ve diğer (Şekil 2B) içinde recoding teli yerleştirin. NOT: ΔV Cal pozitif (Adım 6.4 ve 7.2) ise tellerin kutuplarının doğru. </li><li> Kayıt odasına (Şekil 1B) ve bir karıştırma çubuğu olan bir karıştırma plakası üzerinde odaları yerleştirin. Karıştırma çubuğu elektrot temas etmediğinden emin olun. </li><li> Bir agar köprü (Şekil 2B) (100 mM KCI ve% 2 agar) ile ölçme cihazı. 100 mM KCl Agar köprüleri saklayın. </li&gt; </ol> Cl 4. hazırlanması - Elektrot <ol><li> Eski ve -possibly- çalışmayan pH elektrodu alın. Tamamen gümüş teller ortaya çıkarmak için cam kaplama kırın. </li><li> Dikkatlice neşter bıçak kullanarak gümüş teller kaplama çıkarın. H2, O ve EtOH bol miktarda kullanarak telleri temizleyin. </li><li> Teller kadar FeCl3 (ya da ağartıcı) bir doymuş çözeltisi içinde yer AgCİ muntazam bir koyu kaplama ile kaplanmıştır. </li></ol> Tek tabakalı veziküller 5. N- <ol><li> deneyler önceki gece, dış tamponu, 15 ml (1 mM KCI, SO 4, 25 mM sitrik asit, pH 4.5, 150 mM K 2), hafif sallama ile (kımıldamam) içinde Sephadex G-50 taneleri 1 g kabarma Kullanmadan önce oda sıcaklığında en az 3 saat. Her bir deney, şişmiş boncuk ~ 3 ml gerektirir. En fazla birkaç gün 4 ° C&#39;de şişmiş boncuk tutun. </li><li> Lipozomlar oda sıcaklığında çözülmesi ederken, her sütun ~ 3 m dökmekşişmiş G-50 taneleri l. Oda sıcaklığında yerçekimi akışı ile onları kuru edelim; Bu genellikle 1-2 dakika sürer. </li><li> 0.4 m, teflon kesme kullanarak Mini-ekstruderinden proteoliposomes 11 kes ekstrude tek lamelli kesecikler hazırlanır. </li><li> Plastik yuvarlak dipli bir tüp içinde sütun yerleştirin. Fazla çözüm kaldırmak için sütunları Spin. Bir klinik santrifüj cihazında 1400 x g&#39;de 20-30 saniye santrifüjleyin. </li><li> 13 x 100 mm cam tüpe akış yoluyla yer sütun atın ve kolona ekstrüde veziküller 100 ul ilave edin. Bir klinik santrifüj cihazında 500 xg&#39;de 1 dakika için sütun dönerler. </li><li> Aşama 6.5 kayıt odasına eklenecek akış yoluyla ve ~ 200 ul toplayın. </li></ol> 6. akış ölçümü <ol><li> Referans (toprak) bölmesi içinde 100 mM KCl 2 ml ve dış tampon 1.8 ml yerleştirin (1 mM KCI, 150 mM K 2 SO 4, 25 mM sitrik asit, pH 4.5), kayıt odasına. İç arasındaki hafif ozmotik dengesizlikve dış çözümler etkilemez </li><li> Satın alma programını başlatın. Bu birkaç dakika sürebilir, temel muvazene edelim. </li><li> Sinyal istikrarlı bir temel (lipozomlar hazır ve sütunlar kurumaya) ulaştığında, (Şekil 3A) kaydetmeye başlayın. </li><li> Sistem (Şekil 2A) kalibre edilmesi için bir 10 mM KCI çözeltisi 15 ul ekle. </li><li> Lipozomlar ekleyin. Proteoliposomes (Şekil 3A) - küçük atlama nedeniyle dış Cl eksik çıkarılması için görünür olabilir. Bazal stabilize kadar bekleyin. </li><li> Valinomycin ekleyin (1 mg EtOH / ml 1 ul) akış (Şekil 3A) gerçekleştirmek için kullanılır. </li><li> Akış zamanı kursu bu yaylalar kadar (Şekil 3A) çalıştıralım. </li><li> Tüm lipozomlar (Şekil 3A), çözünmesi için dış tamponu içinde hazırlanmış 1.5 M β-oktilglükozit (β-RG) 40 ul ekle. Kaydı bitirmek. </li></ol> 7. Veri Analysis <ol><li> Bir ASCII veya metin biçiminde gelen izleme dosyası dışa ve seçim analizi programına ithal. </li><li> Aşağıdaki noktaları (Şekil 3A) Gerilim ölçün: Kaydın başlangıcında (V 0); Ayarlama sinyali (V cal) ilave edildikten sonra; Lipozomlar (V lipo) ilave edildikten sonra; Efflux (V kanat) sonunda; Deterjan (V tot) ilave edildikten sonra; Temel gerilimler böylece V 0 = 0. </li><li> Deneysel değerini belirlemek için, Nernst-Plank denklemi kullanın <img alt="Denklem 3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" /> ölçülerek <img alt="Denklem 4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> Nerede Cilt Cal ul kalibrasyon nabız hacmi deneyi ifade başında odası hacmi, 1.800 olduğunu1; - kalibrasyon nabız ve mM dış tampon konsantrasyonları ve ΔV cal kalibrasyon darbesi sonrasında ölçülen mV atlama l [CI] cal / Cl vardır. NOT: α Bu ampirik belirlenmesi üç amaca hizmet eder: sistem düzgün yanıt olmasını sağlar, deneyler arasındaki enstrümanın tepki tutarlılık ölçüsü sunuyor yanı sıra hiçbir hata çözüm yapımında yapılmış olduğunu kontrol etmek. </li><li> Aşağıdaki gibi her adımdan sonra Cl konsantrasyonları hesaplayın: <img alt="Denklem 5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="Denklem 6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="Denklem 7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> faktör 3/400 2000 ul nihai hacim içinde 15 ul kalibrasyon darbe seyrelmesi gelir. </li><li> CalculHer adımda, fin ΔCl lipo / / tot de [Cl] değişiklikleri yedik. </li><li> Ile Cl (t) v (t) dönüştürme <img alt="Denklem 8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> Doğrudan kritik noktalarda [CI] değerlerini belirlemek ve bir iç kontrol olarak hesaplanan değerler bunları karşılaştırmak. </li><li> Iz Normale Cl rel = 0 lipo ve Cl rel tot = 1 böylece <img alt="Denklem 9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li> Aşağıdaki denkleme akış süresi kursu Fit: <img alt="Denklem 10" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> Deneysel olarak Cl ölçülerek belirlenir sızıntısı - - L Cl oranı olduğu durumda, aynı koşullar ve τ hazırlanan proteinsiz lipozomlardan akışını işleminin zaman sabitidir. </li><li> V, ilk hız hesaplayın: Nerede ΔCl fin milimolar ifade ve V ul odasının hacmi olduğunu. </li><li> Taşıma hızı / iletkenlik hesaplayın <img alt="Denklem 12" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> P P / L olduğu durumda, molekül ağırlığı aktif kompleks molekül ağırlığı kDa olarak ifade edilen </li></ol>

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G., del M&#225;rmol, ., J, R., MacKinnon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+of+the+Human+K2P+TRAAK,+a+Lipid-+and+Mechano-Sensitive+K++Ion+Channel.">Crystal Structure of the Human K2P TRAAK, a Lipid- and Mechano-Sensitive K+ Ion Channel.</a> Science. 335, 436-441 (2012).</li><li>Jayaram, H., Robertson, J. L., Wu, F., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+slow+CLC+Cl-/H++antiporter+from+a+cyanobacterium.">Structure of a slow CLC Cl-/H+ antiporter from a cyanobacterium.</a> Biochemistry. 50, 788-794 (2011).</li></ol>

The authors declare no competing financial interests.

Saflaştırılmış anyon-seçici kanallar veya lipozomlar içinde yeniden taşıyıcıları aracılığı ile ulaşım - Biz detaylı Cl ölçmek için protokol tanımlanmıştır. CLC-EC1 eşanjöre - kullanılan örnek Prokaryotik H + / CI oldu. Söz konusu iyon için uygun biri ile AgCİ elektrot: Ancak, bu yöntemin hali hazırda ligandlar 12,13,15, gerilim 11,12, veya Ag değiştirerek farklı anyonik seçicilik 15,16 spor geçitleri kanalları çal...

Son iki aşırı ifade eden ve zar nakil proteinleri saflaştırmak için yeteneği önemli ölçüde artmıştır yıllarda: iyon kanalları, birincil ve ikincil taşıyıcıları rutin heterolog sentezleme sistemleri ile olduğu gibi, doğal kaynaklardan saflaştırılır. Bu proteinlerin istikrar, ifade izlemek geliştirmek ve çıkarma kolaylaştırmak ve geliştirmek için yeni yaklaşımlar sürekli 1-5 geliştirilmektedir. Bu teknolojik devrimler da, kendi işlevinin yapısal üsleri anlayışımızı gelişmiş, membran proteinlerinin atom düzeyinde yapısal bilgi patlamasını tetikleyen vesile olmuştur. Buna karşılık, bizim yetenek bazı durumlarda yüksek çözünürlüklü yapısal bilgiler, böylece kantitatif yapı-tabanlı tahminler sınamak için yeteneğini sınırlayan, nitel verilerle fonksiyonel eşlik böylece, aynı oranda artmadı saflaştırılmış proteinlerin fonksiyonel özelliklerini incelemek için. Bu nedenle, gelişme içerisindenicel ve genellenebilir fonksiyonel testlerin t membran proteini fonksiyonu mekanik temellerinin aydınlatılması yönünde önemli bir adımdır. Kanal, taşıyıcı - Burada kantitatif saflaştınldı ve yeniden Cl anahtar işlevsel özelliklerini belirlemek için kullanılabilecek bir akış deneyi tarif eder. tahlil altında yatan ilkeler taşıma sistemlerinin çeşitli yanı sıra sigara iyon-taşıma proteinlerine jeneralize olabilir. Kanal / büyük Cl mevcudiyetinde taşıyıcıları - - Lipozomlar saflaştırılmış Cı ile yeniden degrade (Şekil 1A, B). Cı - akış bizim durumumuzda, karşı-iyon akışının izin vermek için bir iyonofor ilave edilerek başlatılır K + iyonoforu Valinomycin, Cl tarafından kurulan gerilim şant olan - gradyeni ve K denge potansiyeline ilk membran potansiyeli ayarlanmış + 6,7. T olmadanBir transmembran potansiyeli nesil tarafından engellenir gibi akış oluşur, - o anlamlı net Cl iyonofor&#39;un. - efflux, f 0, aktif protein içermeyen lipozomlar fraksiyonu τ, Cl zaman sabiti: Veri kantitatif iki ölçülebilir parametreler (Şekil 1C) ile tarif edilir. Τ ve f 0 yekpare Cı kaynaktan - taşıma hızı, aktif protein fraksiyonu ve aktif kompleks moleküler kütlesi 8 elde edilebilir. Bilinen yapısı ve fonksiyonu değiştirici - tekniği CLC-EC1, iyi karakterize CLC-tipi H + / Cl ile yeniden proteoliposomes kullanarak burada gösterilmektedir. Bu tahlil kolaylıkla aktivite gerilimi ve / veya ligandların varlığına bağlıdır farklı iyonik seçicilik veya kanallar veya nakliyecilere genelleştirilmiş. Bundan başka, bu deney, küçük moleküller, doğrudan yeniden protein etkileyip etkilemediğini belirlemek için kullanılabilir,Bu bileşiklerin ve nasıl membran bileşimi veya lipid-modifiye reaktifler yeniden kanalların ve taşıyıcıların fonksiyonunu etkileyebilir etkilerini ölçmek için.

<table><tbody><tr><td>Liposomicator, Avanti Polar Lipids Inc.</td><td>&amp;nbsp;Avanti Polar Lipids Inc.</td><td>610200</td><td></td></tr><tr><td>IEC Centra CL2 Benchtop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Orion Research Model 701A Digital pH-mV meter</td><td></td><td></td><td>These can be found on Ebay.</td></tr><tr><td>Non-functional pH probe</td><td></td><td></td><td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td></tr><tr><td>DI-710 Data Logger</td><td>DATAQ instruments</td><td></td><td></td></tr><tr><td>WinDAQ acquisition software</td><td>DATAQ instruments</td><td></td><td></td></tr><tr><td>Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td><td>Pierce (Thermo Scientific)</td><td>29922</td><td></td></tr><tr><td>KIMAX Culture Tubes, Disposable, Borosilicate Glass</td><td>Kimble Chase</td><td>73500-13100</td><td></td></tr><tr><td>Extruder Set With Holder/Heating Block</td><td>Avanti Polar Lipids Inc.</td><td>610000</td><td></td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr></tbody></table>

a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

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.

Değiştirici, lipozomlar içinde yeniden - saflaştırılmış CLC-EC1, bir prokaryotik CLC-tipi H + / Cl aracılığı taşıma - Biz detaylı ve sağlam Cl ölçmek için protokol açıklar. Deneyin şematik bir temsili Şekil 3 &#39;de gösterilmiştir, saflaştırılmış CLC-EC1 ve yüksek iç Cl ihtiva ile yeniden Proteoliposomes -., Düşük Cl ihtiva eden bir banyo çözeltisine daldırıldığı -. Bu koşullar, net Cl altında -</sup...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

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.

Bir Proteoliposome Tabanlı effluks Deney Cl Tek-molekül Properties belirleme<sup&gt; -</sup&gt; Kanallar ve Nakliyeciler

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 ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

9.8K Görüntüleme.  Weill Cornell Medical College. 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.

Video: A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Kolesterol akışı Testi

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

Tıp

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Modelleri ve Hücresel Engeller Arasında İlaç Taşıyıcı Sistemlerin Transport Değerlendirmesinde Yöntemleri

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Biyomühendislik

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton pompalama membran enzimler elektrokimya ve floresan mikroskopisi kullanılarak incelenmesi için tek lipozom ölçümleri

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Biyokimya

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters.

Katı Destekli Membran (SSM) Tabanlı Elektrofizyoloji ile Taşıyıcı Hedefli inhibitör nanobodies seçimi

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidin tabanlı Floresan Testi; çift katlı lipid Özellikleri değiştirmek için Küçük Moleküller Potansiyel Belirleme

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Biyoloji

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Bireysel Lipozomlar Arasındaki Bileşimsel Homojenliği Saptamak için Kantitatif Floresan Mikroskopesi Bazlı Tek Lipozom Testi

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

İçte düzeltici Potasyum Kanallarının Küçük moleküllü Modülatörler için Yüksek throughput Eleme

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

Pseudomonas aeruginosa itibaren MexAB dışa atım pompa in vitro olarak incelenmesi

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membran Transport Tek Protein çözünürlükte Oldukça Paralel nanopore çip Sistemi Analiz Süreçleri

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Bir Proteoliposome Tabanlı effluks Deney Cl Tek-molekül Properties belirleme

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