Questo lavoro è stato finanziato da una sovvenzione da National Heart, Lung, and Blood Institute (NIH R01-HL089564) per KLB e pre-dottorato dalla American Heart Association (09PRE2260209) e Arthur J. Schmitt Fondazione BKM.

<ol>
	<li>Passmore, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNQ/M+Currents+in+Sensory+Neurons:+Significance+for+Pain+Therapy.">KCNQ/M Currents in Sensory Neurons: Significance for Pain Therapy.</a> J. Neurosci. 23, 7227-7236 (2003).</li><li>Falloon, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+small+artery+sensitivity+and+morphology+in+pressurized+and+wire-mounted+preparations.">Comparison of small artery sensitivity and morphology in pressurized and wire-mounted preparations.</a> Am. J. Physiol. Heart Circ. Physiol. 268, H670-H678 (1995).</li><li>Dunn, W. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+big+endothelin-1+in+comparison+with+endothelin-1+on+the+microvascular+blood+flow+velocity+and+diameter+of+rat+mesentery+in+vivo.">Effects of big endothelin-1 in comparison with endothelin-1 on the microvascular blood flow velocity and diameter of rat mesentery in vivo.</a> Microvasc. Res. 72, 108-112 (2006).</li><li>Altura, B. 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. 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Nessun conflitto di interessi dichiarati.

I metodi e gli approcci sperimentali descritti qui sono piuttosto robusti e in grado di produrre risultati chiari e riproducibili se applicato con meticolosa attenzione ai dettagli. Buone registrazioni elettrofisiologiche e costrizione / dilatazione dei segmenti arteriosi dipendono la salute delle cellule e segmenti di arteria, rispettivamente. Preparazioni di cellule può variare da un giorno all&#39;altro, anche utilizzando lo stesso protocollo. Le soluzioni di isolamento può essere utilizzato per un massimo di 2 set...

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Cloruro di sodio </td><td> Sigma </td><td> S5886 </td><td> Soluzione dissezione: 145 Soluzione bagno per Elettrofisiologia *: 140 Soluzione interna per elettrofisiologia: 10 Soluzione di isolamento per miociti *: 140 Soluzione bagno per myography pressione: 145 Soluzione Lumen per myography pressione: 145 </td></tr><tr><td> Cloruro di potassio </td><td> Sigma </td><td> P5405 </td><td> Soluzione dissezione: 4.7 Soluzione bagno per Elettrofisiologia *: 5,36 Soluzione interna per elettrofisiologia: 135 Soluzione di isolamento per miociti *: 5,36 Soluzione bagno per myography pressione: 4.7 Soluzione Lumen per myography pressione: 4.7 </td></tr><tr><td> Potassio EGTA </td><td> Sigma </td><td> E4378 </td><td> Soluzione interna per elettrofisiologia: 0,05 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H9136 </td><td> Soluzione bagno per Elettrofisiologia *: 10 Soluzione interna per elettrofisiologia: 10 Soluzione di isolamento per miociti *: 10 </td></tr><tr><td> Fosfato bibasico </td><td> Sigma </td><td> S5136 </td><td> Soluzione di isolamento per miociti *: 0,34 </td></tr><tr><td> Idrogeno fosfato di potassio </td><td> Sigma </td><td> P5655 </td><td> Soluzione di isolamento per miociti *: 0,44 </td></tr><tr><td> Cloruro di magnesio </td><td> Sigma </td><td> M2393 </td><td> Soluzione bagno per Elettrofisiologia *: 1.2 Soluzione interna per elettrofisiologia: 1 Soluzione di isolamento per miociti *: 1.2 </td></tr><tr><td> Cloruro di calcio </td><td> Sigma </td><td&gt; C7902 </td><td> Soluzione bagno per Elettrofisiologia *: 2 Soluzione di isolamento per miociti *: 0,05 </td></tr><tr><td> Fosfato di sodio </td><td> Fisher Scientific </td><td> BP331-1 </td><td> Soluzione dissezione: 1.2 Soluzione bagno per myography pressione: 1.2 Soluzione Lumen per myography pressione: 1.2 </td></tr><tr><td> Solfato di magnesio </td><td> Sigma </td><td> M2643 </td><td> Soluzione dissezione: 1,17 Soluzione bagno per myography pressione: 1.17 Soluzione Lumen per myography pressione: 1.17 </td></tr><tr><td> MOPS </td><td> Fisher Scientific </td><td> BP308 </td><td> Soluzione dissezione: 3 Soluzione bagno per myography pressione: 3 Soluzione Lumen per myography pressione: 3 </td></tr><tr><td> L&#39;acido piruvico </td><td> Sigma </td><td> P4562 </td><td> Soluzione dissezione: 2 Bath soluzionePressione myography: 2 Soluzione Lumen per myography pressione: 2 </td></tr><tr><td> EDTA diidrato </td><td> La ricerca Organics </td><td> 9572E </td><td> Soluzione dissezione: 0.02 Soluzione bagno per myography pressione: 0.02 Soluzione Lumen per myography pressione: 0.02 </td></tr><tr><td> D-Glucosio </td><td> Sigma </td><td> G7021 </td><td> Soluzione dissezione: 5 Soluzione bagno per Elettrofisiologia *: 10 Soluzione interna per elettrofisiologia: 20 Soluzione di isolamento per miociti *: 10 Soluzione bagno per myography Pressione: 5 Soluzione Lumen per myography Pressione: 5 </td></tr><tr><td> Albumina di siero bovino </td><td> Sigma </td><td> A3912 </td><td> Soluzione dissezione: 1% Soluzione Lumen per myography pressione: 1% </td></tr><tr><td> pH </td><td></td><td></td><td> Dissezione Solution: 7.4 Soluzione bagno per Elettrofisiologia *: 7.3 Soluzione interna per elettrofisiologia: 7.2 Soluzione di isolamento per miociti *: 7.2 Soluzione bagno per myography pressione: 7.4 Soluzione Lumen per myography pressione: 7.4 </td></tr><tr><td> Osmolarità </td><td></td><td></td><td> Soluzione dissezione: 300 Soluzione bagno per Elettrofisiologia *: 298 Soluzione interna per elettrofisiologia: 298 Soluzione di isolamento per miociti *: 298 Soluzione bagno per myography pressione: 300 Soluzione Lumen per myography pressione: 300 </td></tr><tr><td></td><td></td><td></td><td> * 11 </td></tr><tr><td></td><td></td><td></td><td> Tabella 1. Componenti di soluzioni utilizzate nell&#39;esperimento. </td></tr></tbody></table>

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

Contrazione o rilassamento delle cellule muscolari lisce all&#39;interno delle pareti delle arterie di resistenza determina il diametro dell&#39;arteria e controlla quindi il flusso di sangue attraverso il vaso e contribuisce alla pressione arteriosa sistemica. Il processo di contrazione è regolato principalmente dalla concentrazione di calcio citosolico ([Ca 2 +] cyt), che è a sua volta controllata da una serie di trasportatori e canali ionici. I canali ionici sono intermedi comuni vie di trasduzione del segnale attivati ​​da ormoni vasoattivi per effettuare vasocostrizione o vasodilatazione. E i canali ionici sono spesso presi di mira da agenti terapeutici intenzionalmente (es. calcio antagonisti utilizzati per indurre vasodilatazione e la pressione sanguigna più bassa) o involontariamente (ad esempio per indurre indesiderati effetti collaterali cardiovascolari). KV7 (KCNQ) voltaggio-attivati ​​i canali del potassio sono stati recentemente implicati come importante targ fisiologici e terapeuticiets per la regolazione della contrazione della muscolatura liscia. Per chiarire i ruoli specifici dei KV7 canali sia trasduzione del segnale fisiologico e nelle azioni di agenti terapeutici, bisogna studiare come la loro attività è modulata a livello cellulare e valutare il loro contributo nell&#39;ambito dell&#39;arteria intatta. Le arterie mesenteriche ratto forniscono un utile sistema modello. Le arterie possono essere facilmente sezionato, puliti del tessuto connettivo, e utilizzato per preparare isolate miociti arteriosi patch clamp per elettrofisiologia, o incannulata e pressione per misure di vasocostrittori / vasodilatatore risposte fisiologiche in condizioni relativamente. Qui descrivere i metodi utilizzati per entrambi i tipi di misure e di fornire alcuni esempi di come il disegno sperimentale possono essere integrati per fornire una comprensione più chiara dei ruoli di questi canali ionici nella regolazione del tono vascolare.

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

Misure di KV7 (KCNQ) canale attività di potassio in miociti isolati arteriosi (utilizzando tecniche elettrofisiologiche di patch clamp) in parallelo con misure di constrictor / dilatatore risposte (usando myography pressione) può rivelare importanti informazioni sul ruolo di KV7 canali in fisiologia vascolare muscolatura liscia e farmacologia.

Esplorare arteriosa Muscolo liscio KV7 Funzione canale del potassio con morsetto Elettrofisiologia Patch e Myography pressione

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

15.1K Views.  Loyola University Chicago. Misure di KV7 (KCNQ) canale attività di potassio in miociti isolati arteriosi (utilizzando tecniche elettrofisiologiche di patch clamp) in parallelo con misure di constrictor / dilatatore risposte (usando myography pressione) può rivelare importanti informazioni sul ruolo di KV7 canali in fisiologia vascolare muscolatura liscia e farmacologia.

Video: 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.

Metodi per la registrazioni Morsetto capacità patch dal Calyx

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

Ricerca

Biologia

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.

Pressione lucidatura Pipette per una migliore patch-clamp di registrazione

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.

Focale Ca 2 + Rilevamento transitoria nel muscolo liscio

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.

Isolamento di cellule umane ombelicale muscolatura liscia arteriosa (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.

Giant Preparazione liposomi per Immagini e di patch-clamp elettrofisiologia

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.

Valutare Murino Resistenza Funzione Arteria Utilizzando myography Pressione

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 canale Cell-allegato patch-clamp registrazione

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.

Ricostituzione di una proteina transmembrana, la tensione gated canali ionici, KvAP, in Giant unilamellari vescicole per la microscopia e Patch Clamp Studies

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.

Misura di Smooth funzione muscolare nei isolato carta igienica-applicazioni Farmacologia Research

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

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.