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

1. Préparation des lipides <ol><li> Aliquoter les lipides quantité désirée dans un tube de verre clair. Tu Vois. coli extrait lipidique polaire, mais la plupart des compositions lipidiques peuvent être utilisés. Si les lipides sont sous forme de poudre, les remettre en suspension dans du chloroforme à une concentration de 20 mg / ml. Sécher les lipides à température ambiante sous N2 gaz jusqu&#39;à ce que tout le solvant se est évaporé. </li><li> Reprendre les lipides dans le pentane et le sécher à nouveau un flux régulier de gaz N 2 pour éliminer les traces de restes de chloroforme. Sec jusqu&#39;à ce que tout le solvant se est évaporé. Bien que le séchage, faire tourner doucement le tube de façon que le lipide est distribué dans un film uniforme couvrant le tiers inférieur du tube plutôt que d&#39;une formation d&#39;une masse dense à la base. </li><li> Ajouter le tampon de reconstitution contenant un détergent (300 mM de KCl, 50 mM d&#39;acide citrique, 25 mM de K 2 HPO 4, à pH 4,5, CHAPS 35 mM) pour dissoudre les lipides. Utiliser d&#39;autres détergents doux pour cette étape si la protéine est mal tolérants à CHAPS. </li><li> ResuspenD les lipides à la clarté en utilisant un appareil à ultrasons à bain d&#39;eau. Plonger le tube dans le centre du bain de sonicateur de l&#39;eau à la puissance maximale à court (30 sec-1 min) avec des impulsions de courte durée (30 sec-1 min) pauses. La solution doit devenir clair. NOTE: Le dernier degré de clarté atteint dépend de la composition lipidique spécifique utilisé. Une certaine variation de lot à lot dans cette étape, même parmi les lipides nominalement identiques, est également possible. Trouble résiduel est dû à la diffusion de lumière par les grosses particules dans la suspension, qui peuvent être éliminés par centrifugation. </li><li> Incuber les lipides remises en suspension à température ambiante pendant 20 à 60 min. </li></ol> 2. Formation protéoliposome REMARQUE: Plusieurs stratégies peuvent être employées pour introduire la protéine solubilisée par un détergent dans des liposomes. Pour CLC-ec1 dialyse fonctionne bien et est donc la méthode de choix 6,9,10. <ol><li> Ajouter la protéine purifiée de la protéine souhaitée à des lipides (P / L) rapport (exprimé en pg o<html> f protéine / mg de lipide). Le choix du rapport P / L dépend du but de l&#39;expérience. <ol><li> Si le but est d&#39;évaluer l&#39;activité de la protéine d&#39;intérêt, à haute reconstituer P / L&#39;s pour maximiser le signal, chaque liposome contient des copies multiples de la protéine. Pour quantifier l&#39;activité et / ou la vitesse de transport unitaire de la protéine, reconstituer dans un régime à basse dilution Poisson P / L&#39;s, de sorte que chaque vésicule contient en moyenne une protéine. Voir l&#39;étape 7 et discussion pour une analyse plus approfondie de l&#39;utilisation de chaque régime. </li></ol></li><li> Pour déterminer les valeurs P / L optimales pour les différents schémas, effectuer un titrage complet de l&#39;activité en fonction de la concentration en protéine 8,11-13. Toutefois, pour toute protéine pour laquelle le poids moléculaire (MW, exprimée en kDa) de l&#39;unité active est connu dans le P / L valeurs suivantes peuvent être utilisés comme des estimations approximatives initiales: pg &quot;/&gt; <img alt="Equation 2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li> Charger le mélange de protéines et de lipides dans un tube de dialyse pré-mouillée ou une cassette. Placer le dispositif de dialyse dans un bêcher contenant 500-1,000x le volume du tampon de reconstitution lipide (par exemple, une mémoire tampon pour L 1 ml de lipide) remise en suspension sous agitation douce. <ol><li> Changer tampon 3-4 fois à intervalles&gt; 3 h. A chaque changement de solution, laver la cassette / tube et le bécher avec de l&#39;eau distillée et remplir le bécher avec le tampon de reconstitution frais. Inclure 1-2 O N / intervalles pour le changement de solution. Le nombre exact et la durée des changements de la solution dépend de la lipide exacte et les détergents utilisés. </li></ol></li><li> Après la dernière étape est terminée, retirez les liposomes de la cuve de dialyse, les aliquoter dans des tubes et flash les congeler dans un liquide N 2 ou gel à -80 ° C. </li></ol> 3. Enregistrement Set-up &lt;p class = &quot;jove_content&quot;&gt; NOTE: L&#39;enregistrement mis en place (figure 2A) se compose de deux chambres (cylindres à fond plat, ~ 3-4 ml de volume), un Cl - (voir ci-dessous) électrode, un pH-mètre avec un analogue ou sortie numérique électrique, un numériseur et un ordinateur avec un logiciel d&#39;acquisition appropriée. <ol><li> Connecter le Cl - électrode de pH-mètre, la sortie de l&#39;appareil de mesure du pH au numériseur qui est relié à l&#39;ordinateur. Placer l&#39;électrode de référence dans une chambre de recodage et le fil dans l&#39;autre (figure 2B). NOTE: La polarité des fils est correct si AV Cal (voir l&#39;étape 6.4 et 7.2) est positif. </li><li> Placez les chambres sur une plaque d&#39;agitation avec un barreau d&#39;agitation dans la chambre d&#39;enregistrement (figure 1B). Assurez-vous que la barre d&#39;agitation ne touche pas l&#39;électrode. </li><li> Raccorder les chambres en utilisant un pont d&#39;agar (figure 2B) (100 mM de KCl et 2% d&#39;agarose). Conserver les ponts Agar dans 100 mM de KCl. </li&gt; </ol> 4. Préparation du Cl - électrode <ol><li> Prenez un vieux et -possibly- électrode de pH non-fonctionnement. Briser le revêtement de verre pour révéler complètement les fils d&#39;argent. </li><li> Retirez délicatement le revêtement des fils d&#39;argent en utilisant une lame de scalpel. Nettoyez les fils à l&#39;aide de grandes quantités de H 2 O et EtOH. </li><li> Placer dans une solution saturée de FeCl 3 (ou eau de Javel) jusqu&#39;à ce que les fils sont recouverts d&#39;un manteau sombre uniforme de AgCl. </li></ol> 5. Préparation de vésicules unilamellaires <ol><li> La nuit avant les expériences, gonfler 1 g de Sephadex G-50 perles dans 15 ml de tampon externe (1 mM de KCl, 150 mM de K 2 SO 4, l&#39;acide citrique 25 mM, pH 4,5) en agitant doucement (Ne pas remuer) pour au moins trois heures à température ambiante avant utilisation. Chaque expérience nécessite ~ 3 ml de perles gonflées. Gardez les perles gonflées à 4 ° C pendant quelques jours au plus. </li><li> Alors que les liposomes sont décongélation à température ambiante, verser dans chaque colonne ~ 3 ml de gonflées G-50 perles. Laissez-les sécher par gravité à la température ambiante; cela prend habituellement 1-2 min. </li><li> Préparer vésicules unilamellaires par extrusion des protéoliposomes 11 fois à travers un mini-extrudeuse en utilisant un seuil de coupure de 0,4 m de Teflon. </li><li> Placer la colonne dans un tube à fond rond en matière plastique. Faites tourner les colonnes pour éliminer l&#39;excès de solution. Centrifuger pendant 20 à 30 secondes à 1400 xg dans une centrifugeuse clinique. </li><li> Jeter accréditives, lieu colonne dans un tube de verre 13 x 100 mm et ajouter 100 pi des vésicules extrudées à la colonne. Spin colonne pendant 1 min à 500 g dans une centrifugeuse clinique. </li><li> Recueillir ~ 200 pi d&#39;accréditives qui seront ajoutés à la chambre d&#39;enregistrement à l&#39;étape 6.5. </li></ol> 6. Efflux mesure <ol><li> Placer 2 ml de 100 mM de KCl dans la référence (masse) chambre et 1,8 ml du tampon externe (1 mM de KCl, 150 mM de K 2 SO 4, l&#39;acide citrique mM 25 à pH 4,5) à la chambre d&#39;enregistrement. Le léger déséquilibre osmotique entre l&#39;interneet des solutions externes ne affecte pas </li><li> Démarrez le programme d&#39;acquisition. Laissez la ligne de base équilibrer, cela peut prendre quelques minutes. </li><li> Une fois le signal atteint une ligne de base stable (les liposomes sont prêts et les colonnes sèches), commencer l&#39;enregistrement (figure 3A). </li><li> Ajouter 15 ul d&#39;une solution 10 mM de KCl pour calibrer le système (figure 2A). </li><li> Ajouter liposomes. Un petit saut pourrait être visible en raison de l&#39;élimination incomplète de Cl externe - des protéoliposomes (figure 3A). Attendez que la ligne de base se stabilise. </li><li> Ajouter valinomycine (1 ul à 1 mg / ml dans EtOH) pour initier l&#39;efflux (figure 3A). </li><li> Laissez l&#39;efflux suivre son évolution dans le temps jusqu&#39;à ce qu&#39;il plateaux (figure 3A). </li><li> Ajouter 40 ul de 1,5 M β-octylglucoside (β-OG) préparé dans le tampon externe pour dissoudre tous les liposomes (figure 3A). Terminez l&#39;enregistrement. </li></ol> 7. A donnéesnalyse <ol><li> Exportez le fichier de trace à partir dans un format ASCII ou texte et l&#39;importer dans le programme d&#39;analyse de choix. </li><li> Mesurer la tension aux points suivants (figure 3A): Au début de l&#39;enregistrement (V 0); Après addition de l&#39;impulsion d&#39;étalonnage (V cal); Après addition des liposomes (V lipo); A la fin de l&#39;écoulement (V fin); Après addition de détergent (V tot); Baseline les tensions de sorte que V 0 = 0. </li><li> Utilisez l&#39;équation de Nernst-Planck pour déterminer la valeur expérimentale de <img alt="Équation 3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" /> en mesurant <img alt="Equation 4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> Où Vol Cal est le volume de l&#39;impulsion d&#39;étalonnage dans ul, 1800 est le volume de la chambre au début de l&#39;expérience exprimée en1; l, [Cl] cal / en sont les Cl - les concentrations de l&#39;impulsion de l&#39;étalonnage et de la mémoire tampon externe mM et AV cal est le saut en mV mesurée après l&#39;impulsion d&#39;étalonnage. NOTE: Cette détermination empirique de α répond à trois objectifs: il se assure que le système répond correctement, offre une mesure de la cohérence de la réponse de l&#39;instrument entre les expériences ainsi que pour vérifier qu&#39;aucun erreurs ont été commises dans la fabrication de la solution. </li><li> Calculer les concentrations en Cl après chaque étape comme suit: <img alt="Equation 5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="Equation 6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="Equation 7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> Le facteur 3/400 vient de la dilution de l&#39;impulsion de calibrage de 15 ul dans un volume final de 2000 pl. </li><li> Calculmangé les changements dans [Cl] à chaque étape, ΔCl lipo / FIN / tot. </li><li> Autre V (t) Cl (t) avec <img alt="Equation 8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> Directement déterminer les [CL] valeurs aux points critiques et de les comparer aux valeurs calculées comme un contrôle interne. </li><li> Normaliser la trace de sorte que la lipo Cl rel = 0 et Cl rel tot = 1 <img alt="Equation 9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li> Monter le cours de temps d&#39;écoulement à l&#39;équation suivante: <img alt="Equation 10" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> Où L est le taux de Cl - fuite qui est déterminé expérimentalement en mesurant Cl - efflux de liposomes exempts de protéines préparés dans les mêmes conditions et τ est la constante de temps du processus. </li><li> Calculer v, la vitesse initiale: Lorsque ΔCl ailette est exprimée en millimoles et V est le volume de la chambre en pi. </li><li> Calculer le taux de transport / conductance <img alt="Equation 12" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> Où p est le rapport P / L, MW est le poids moléculaire du complexe actif exprimée en kDa </li></ol>

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The authors declare no competing financial interests.

Nous avons décrit un protocole détaillé pour mesurer Cl - le transport médié par les canaux ou transporteurs reconstitués dans des liposomes anioniques sélectif purifiés. L&#39;exemple utilisé était le procaryote H + / Cl - Échangeur CLC-CE1. Toutefois, la méthode peut être facilement adapté à l&#39;étude canaux gated par des ligands 12,13,15, 11,12 tension, ou de sport différente sélectivité anionique 15,16 en remplaçant le Ag: AgCl par celle ad...

Dans les deux dernières décennies, la capacité de surexprimer et purifier des protéines de transport membranaire a considérablement augmenté: les canaux ioniques, les transporteurs primaires et secondaires sont maintenant couramment purifiés à partir de systèmes d&#39;expression hétérologues, ainsi que des sources naturelles. De nouvelles approches pour surveiller l&#39;expression, améliorer et faciliter l&#39;extraction et à améliorer la stabilité de ces protéines sont constamment mises au point 1-5. Ces avancées technologiques ont joué un rôle dans le déclenchement de l&#39;explosion de l&#39;information structurelle niveau atomique de protéines membranaires qui, à son tour, amélioré notre compréhension des bases structurelles de leur fonction. En revanche, notre capacité à sonder les propriétés fonctionnelles des protéines purifiées ne ont pas augmenté au même rythme, de sorte que dans certains cas, des informations structurelles haute résolution est accompagnée de données fonctionnelles qualitatives, limitant ainsi notre capacité à tester quantitativement prédictions basées sur la structure. Par conséquent, la mist des analyses fonctionnelles quantitatives et généralisables est une étape clé pour l&#39;élucidation des fondements mécanistes de la fonction des protéines de la membrane. Nous décrivons ici un essai d&#39;écoulement qui peut être utilisé pour déterminer quantitativement des propriétés fonctionnelles clés de reconstituées purifiées et Cl - canaux et des transporteurs. Les principes qui sous-tendent l&#39;analyse peuvent être généralisés à une variété de systèmes de transport, ainsi que des protéines de transport non-ioniques. Les liposomes sont reconstitués purifiés avec canal Cl - / transporteurs en présence d&#39;un grand Cl - gradient (Figure 1A, B). Cl - efflux est initiée par l&#39;addition d&#39;un ionophore pour permettre un flux contre-ion, dans notre cas le K + ionophore valinomycine, qui shunter la tension établie par le Cl - gradient et régler le potentiel de la membrane initiale au potentiel de K d&#39;équilibre + 6,7. Sans til ne ionophore Cl nette significative - efflux se produit, comme cela est empêché par la génération d&#39;un potentiel transmembranaire. Les données sont quantitativement décrite par deux paramètres mesurables (figure 1c): τ, la constante de temps de Cl - efflux, et f 0, la fraction de liposomes ne contenant pas de protéine active. De τ et f 0 Cl unitaire - taux de transport, la fraction de protéines actives et la masse moléculaire du complexe actif peuvent être dérivées 8. La technique est illustrée ici en utilisant protéoliposomes reconstituées avec la SIC-CE1, un type bien caractérisé CTC H + / Cl - échangeur de la structure et de la fonction connue. Ce dosage est facilement généralisable à des canaux ou transporteurs à sélectivité ionique ou différent, dont l&#39;activité dépend de la présence de tension et / ou des ligands. En outre, ce test peut être utilisé pour déterminer si petites molécules affectent directement la protéine reconstitué,pour quantifier les effets de ces composés et de la façon dont la composition de la membrane ou des réactifs lipidiques modificateurs affectent la fonction des canaux reconstituées et les transporteurs.

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			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
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			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
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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.

Nous décrivons un protocole détaillé et robuste pour mesurer Cl - le transport médié par la SIC-ec1 purifiée, un type CTC procaryote H + / Cl - échangeur, reconstituée dans des liposomes. Une représentation schématique de l&#39;expérience est illustré à la figure 3 protéoliposomes reconstituées avec la SIC-ec1 purifié et contenant haute Cl interne -. Sont immergés dans une solution de bain contenant faible Cl -. Dans ces conditions ...

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.

Un protéoliposome-Based Assay Efflux pour déterminer les propriétés unique molécule de Cl<sup&gt; -</sup&gt; Chaînes et transporteurs

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

Research

JoVE Journal

Biology

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

9.7K vues.  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.

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

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Méthodes Single Molecule pour surveiller les changements dans les propriétés élastique bicouche

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Recherche

Biologie

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualisation de la réplication d&#39;ADN molécule unique par microscopie à fluorescence

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

Observation directe d&#39;ADN Enzymes réplication via un ADN simple molécule Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Molécules simples à l&#39;imagerie du transport nucléaire

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un test de dépistage fluorescent pour identifier des modulateurs de canaux Girk

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

La combinaison unique molécule manipulation et d&#39;imagerie pour l&#39;étude des interactions protéine-ADN

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Étude de l&#39;ADN Looping par une seule molécule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Mesure et analyse de la production extracellulaire acide pour Déterminer glycolytique Taux

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Le test de fixation du Lactate déshydrogénase — Une méthode Simple et fiable pour déterminer l’activité de séquestration autophagique en vrac dans des cellules de mammifères

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Essai cinétique à molécule unique à haut débit parallèle pour le clivage d’ADN spécifique au site

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...