Ce travail a été financé par une subvention du National Heart, Lung, and Blood Institute (NIH R01-HL089564) pour KLB et pré-doctorales de l&#39;American Heart Association (09PRE2260209) et Arthur J. Schmitt Fondation pour BKM.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-response+relationships+for+arginine+vasopressin+and+synthetic+analogs+on+three+types+of+rat+blood+vessels:+possible+evidence+for+regional+differences+in+vasopressin+receptor+sites+within+a+mammal.">Dose-response relationships for arginine vasopressin and synthetic analogs on three types of rat blood vessels: possible evidence for regional differences in vasopressin receptor sites within a mammal.</a> J. Pharmacol. Exp. Ther. 193, 413-423 (1975).</li><li>Henderson, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasopressin-induced+vasoconstriction:+two+concentration-dependent+signaling+pathways.">Vasopressin-induced vasoconstriction: two concentration-dependent signaling pathways.</a> J. Appl. Physiol. 102, 1402-1409 (2007).</li><li>Mackie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+KCNQ+potassium+channels+as+novel+targets+for+the+control+of+mesenteric+artery+constriction+by+vasopressin,+based+on+studies+in+single+cells,+pressurized+arteries,+and+in+vivo+measurements+of+mesenteric+vascular+resistance.">Vascular KCNQ potassium channels as novel targets for the control of mesenteric artery constriction by vasopressin, based on studies in single cells, pressurized arteries, and in vivo measurements of mesenteric vascular resistance.</a> J. Pharmacol. Exp. Ther. 325, 475-483 (2008).</li><li>Brueggemann, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+selective+cyclooxygenase-2+inhibitors+on+vascular+smooth+muscle+ion+channels+may+account+for+differences+in+cardiovascular+risk+profiles.">Differential effects of selective cyclooxygenase-2 inhibitors on vascular smooth muscle ion channels may account for differences in cardiovascular risk profiles.</a> Mol. Pharmacol. 76, 1053-1061 (2009).</li><li>Brueggemann, L. I., Kaneez, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamp Technique. , (2012).</li><li>Berra-Romani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TTX-sensitive+voltage-gated+Na++channels+are+expressed+in+mesenteric+artery+smooth+muscle+cells.">TTX-sensitive voltage-gated Na+ channels are expressed in mesenteric artery smooth muscle cells.</a> Am. J. Physiol. Heart Circ. Physiol. 289, H137-H145 (2005).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Les méthodes et approches expérimentales décrites ici sont très robustes et peuvent produire des résultats clairs et reproductibles lorsqu&#39;ils sont appliqués avec une attention méticuleuse aux détails. De bons enregistrements électrophysiologiques et la constriction / dilatation des segments artériels sont tributaires de la santé des cellules et des segments d&#39;artère, respectivement. Préparations de cellules peut varier de jour en jour, même en utilisant le même protocole. Solutions d&#39;isolatio...

<table border="1"><tbody><tr><td> Nom </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Chlorure de sodium </td><td> Sigma </td><td> S5886 </td><td> Solution dissection: 145 Solution de bain pour électrophysiologie *: 140 Solution interne pour l&#39;électrophysiologie: 10 Solution d&#39;isolation pour les myocytes *: 140 Solution de bain pour myographie pression: 145 Lumen solution pour myographie pression: 145 </td></tr><tr><td> Le chlorure de potassium </td><td> Sigma </td><td> P5405 </td><td> Solution dissection: 4,7 Solution de bain pour électrophysiologie *: 5,36 Solution interne pour l&#39;électrophysiologie: 135 Solution d&#39;isolation pour les myocytes *: 5,36 Solution de bain pour myographie pression: 4,7 Lumen solution pour myographie pression: 4,7 </td></tr><tr><td> Potassium EGTA </td><td> Sigma </td><td> E4378 </td><td> Solution interne pour l&#39;électrophysiologie: 0,05 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H9136 </td><td> Solution de bain pour électrophysiologie *: 10 Solution interne pour l&#39;électrophysiologie: 10 Solution d&#39;isolation pour les myocytes *: 10 </td></tr><tr><td> Hydrogénophosphate disodique </td><td> Sigma </td><td> S5136 </td><td> Solution d&#39;isolation pour les myocytes *: 0,34 </td></tr><tr><td> Hydrogénophosphate de potassium </td><td> Sigma </td><td> P5655 </td><td> Solution d&#39;isolation pour les myocytes *: 0,44 </td></tr><tr><td> Chlorure de magnésium </td><td> Sigma </td><td> M2393 </td><td> Solution de bain pour électrophysiologie *: 1,2 Solution interne pour l&#39;électrophysiologie: 1 Solution d&#39;isolation pour les myocytes *: 1,2 </td></tr><tr><td> Chlorure de calcium </td><td> Sigma </td><td&gt; C7902 </td><td> Solution de bain pour électrophysiologie *: 2 Solution d&#39;isolation pour les myocytes *: 0,05 </td></tr><tr><td> Phosphate de sodium </td><td> Fisher Scientific </td><td> BP331-1 </td><td> Solution dissection: 1.2 Solution de bain pour myographie pression: 1,2 Lumen solution pour myographie pression: 1,2 </td></tr><tr><td> Sulfate de magnésium </td><td> Sigma </td><td> M2643 </td><td> Solution dissection: 1,17 Solution de bain pour myographie pression: 1,17 Lumen solution pour myographie pression: 1,17 </td></tr><tr><td> MOPS </td><td> Fisher Scientific </td><td> BP308 </td><td> Solution dissection: 3 Solution de bain pour myographie pression: 3 Lumen solution pour myographie pression: 3 </td></tr><tr><td> L&#39;acide pyruvique </td><td> Sigma </td><td> P4562 </td><td> Solution dissection: 2 Solution de bain d&#39;myographie pression: 2 Lumen solution pour myographie pression: 2 </td></tr><tr><td> Dihydraté EDTA </td><td> Biologique Recherche </td><td> 9572E </td><td> Solution dissection: 0,02 Solution de bain pour myographie pression: 0,02 Lumen solution pour myographie pression: 0,02 </td></tr><tr><td> Le D-glucose </td><td> Sigma </td><td> G7021 </td><td> Solution dissection: 5 Solution de bain pour électrophysiologie *: 10 Solution interne pour l&#39;électrophysiologie: 20 Solution d&#39;isolation pour les myocytes *: 10 Solution de bain pour myographie pression: 5 Lumen solution pour myographie pression: 5 </td></tr><tr><td> Sérum-albumine bovine </td><td> Sigma </td><td> A3912 </td><td> Solution dissection: 1% Lumen solution pour myographie pression: 1% </td></tr><tr><td> pH </td><td></td><td></td><td> Dissection Solution: 7,4 Solution de bain pour électrophysiologie *: 7,3 Solution interne pour l&#39;électrophysiologie: 7.2 Solution d&#39;isolation pour les myocytes *: 7,2 Solution de bain pour myographie pression: 7,4 Lumen solution pour myographie pression: 7,4 </td></tr><tr><td> Osmolarité </td><td></td><td></td><td> Solution dissection: 300 Solution de bain pour électrophysiologie *: 298 Solution interne pour l&#39;électrophysiologie: 298 Solution d&#39;isolation pour les myocytes *: 298 Solution de bain pour myographie pression: 300 Lumen solution pour myographie pression: 300 </td></tr><tr><td></td><td></td><td></td><td> * 11 </td></tr><tr><td></td><td></td><td></td><td> Tableau 1. Composants des solutions utilisées dans l&#39;expérience. </td></tr></tbody></table>

exploring arterial smooth muscle kv7 potassium channel function using patch clamp electrophysiology and pressure myography

Contraction ou la relaxation des cellules musculaires lisses dans les parois des artères de résistance détermine le diamètre et commande ainsi l&#39;artère écoulement de sang à travers le vaisseau et contribue à la pression artérielle systémique. Le processus de contraction est régulée par la concentration en calcium cytosolique ([Ca 2 +] cyt), qui est à son tour commandé par une série de transporteurs d&#39;ions et des canaux. Les canaux ioniques sont des intermédiaires communs dans les voies de transduction du signal activées par les hormones vasoactives pour effectuer une vasoconstriction ou une vasodilatation. Et des canaux ioniques sont souvent pris pour cible par des agents thérapeutiques soit intentionnellement (par exemple inhibiteurs calciques utilisés pour induire une vasodilatation et hypotension) ou non (par exemple, pour induire des effets secondaires indésirables cardio-vasculaires). KV7 (KCNQ) canaux potassiques voltage-activés ont récemment été identifié comme targ physiologique et thérapeutique importanteets de régulation de la contraction du muscle lisse. Pour élucider les rôles spécifiques des KV7 canaux à la fois dans la transduction du signal physiologique et dans les actions des agents thérapeutiques, nous devons étudier la façon dont leur activité est modulée au niveau cellulaire ainsi que d&#39;évaluer leur contribution dans le cadre de l&#39;artère intacte. Les artères mésentériques de rats constituent un système modèle utile. Les artères peuvent être facilement disséquées, débarassés des tissus conjonctifs, et utilisé pour préparer des myocytes artériels isolés pour patch clamp électrophysiologie, ou une canule et mis sous pression pour les mesures de vasoconstricteurs / réponses vasodilatateur dans des conditions relativement physiologiques. Nous décrivons ici les méthodes utilisées pour les deux types de mesures et de donner quelques exemples de la façon dont la conception expérimentale peut être intégré à fournir une meilleure compréhension des rôles de ces canaux ioniques dans la régulation du tonus vasculaire.

Watch this Scientific Journal Video about Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography at JoVE.com

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Les mesures de KV7 (KCNQ) l&#39;activité des canaux potassium dans les myocytes isolés des artères (l&#39;utilisation du patch clamp techniques électrophysiologiques) en parallèle avec des mesures de constricteurs / dilatateur réponses (en utilisant myographie pression) peut révéler des informations importantes sur le rôle des canaux KV7 dans la physiologie du muscle lisse vasculaire et pharmacologie.

Explorer muscle lisse artériel KV7 fonction des canaux potassiques à l&#39;aide d&#39;électrophysiologie patch-clamp et myographie pression

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

exploring-arterial-smooth-muscle-kv7-potassium-channel-function-using

Research

JoVE Journal

Biology

15.0K vues.  Loyola University Chicago. Les mesures de KV7 (KCNQ) l'activité des canaux potassium dans les myocytes isolés des artères (l'utilisation du patch clamp techniques électrophysiologiques) en parallèle avec des mesures de constricteurs / dilatateur réponses (en utilisant myographie pression) peut révéler des informations importantes sur le rôle des canaux KV7 dans la physiologie du muscle lisse vasculaire et pharmacologie.

Vidéo: Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Méthodes pour les enregistrements patch clamp Capacité du calice

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Recherche

Biologie

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Enregistrement Patch Clamp des canaux ioniques exprimés dans les oocytes de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Pression de polissage Pipettes Amélioration patch-clamp

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Ca focale 2 + Détection de transitoires dans le muscle lisse

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

L&#39;isolement de l&#39;homme de l&#39;artère ombilicale cellules musculaires lisses (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

Préparation des liposomes de géant pour l&#39;imagerie et de patch-clamp électrophysiologie

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Évaluer murin Fonction Artère résistance à l&#39;aide myographie de pression

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canal cellulaire attaché patch-clamp enregistrement

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstitution d&#39;une protéine transmembranaire, le Ion canal de voltage-dépendants, KvAP, en géant vésicules unilamellaires pour la microscopie et Patch études Clamp

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Mesure de la fonction musculaire lisse dans les tissus isolés Bath-applications à la pharmacologie de recherche

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...