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

10.3791/52281 ◽  
2015 ◽  
Author(s):  
Matthias Garten ◽  
Sophie Aimon ◽  
Patricia Bassereau ◽  
Gilman E. S. Toombes
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Transmembrane Protein ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Voltage Gated

Classification of potassium and chlorine ionic currents in retinal ganglion cell line (RGC-5) by whole-cell patch clamp

2012 ◽  
Vol 29 (6) ◽  
pp. 275-282 ◽  
Author(s):  
SHU-JIE WANG ◽  
LAI-HUA XIE ◽  
BIN HENG ◽  
YAN-QIANG LIU
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Ganglion Cell ◽  
Cell Line ◽  
Ionic Currents ◽  
Retinal Ganglion ◽  
Voltage Gated ◽  
Whole Cell Patch Clamp ◽  
Inwardly Rectifying

AbstractRetinal ganglion cell line (RGC-5) has been widely used as a valuable model for studying pathophysiology and physiology of retinal ganglion cells in vitro. However, the electrophysiological characteristics, especially a thorough classification of ionic currents in the cell line, remain to be elucidated in details. In the present study, we determined the resting membrane potential (RMP) in RGC-5 cell line and then identified different types of ionic currents by using the whole-cell patch-clamp technique. The RMP recorded in the cell line was between −30 and −6 mV (−17.6 ± 2.6 mV, n = 10). We observed the following voltage-gated ion channel currents: (1) inwardly rectifying Cl− current (ICl,ir), which could be blocked by Zn2+; (2) Ca2+-activated Cl− current (ICl,Ca), which was sensitive to extracellular Ca2+ and could be inhibited by disodium 4,4’-diisothiocyanatostilbene-2,2’-disulfonate; (3) inwardly rectifying K+ currents (IK1), which could be blocked by Ba2+; (4) a small amount of delayed rectifier K+ current (IK). On the other hand, the voltage-gated sodium channels current (INa) and transient outward potassium channels current (IA) were not observed in this cell line. These results further characterize the ionic currents in the RGC-5 cell line and are beneficial for future studies especially on ion channel (patho)physiology and pharmacology in the RGC-5 cell line.

Phospholamban spontaneously reconstitutes into giant unilamellar vesicles where it generates a cation selective channel

2016 ◽  
Vol 18 (3) ◽  
pp. 1629-1636 ◽  
Author(s):  
S. Smeazzetto ◽  
F. Tadini-Buoninsegni ◽  
G. Thiel ◽  
D. Berti ◽  
C. Montis
Keyword(s):  
Ion Channel ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Selective Channel ◽  
Cation Selective Channel

Reconstitution of phospholamban in giant unilamellar vesicles and generation of an ion channel.

scholarly journals Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles

PLoS ONE ◽  
2011 ◽  
Vol 6 (10) ◽  
pp. e25529 ◽  
Author(s):  
Sophie Aimon ◽  
John Manzi ◽  
Daniel Schmidt ◽  
Jose Antonio Poveda Larrosa ◽  
Patricia Bassereau ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Voltage Gated

scholarly journals Integrin-Functionalised Giant Unilamellar Vesicles via Gel-Assisted Formation: Good Practices and Pitfalls

2021 ◽  
Vol 22 (12) ◽  
pp. 6335
Author(s):  
Mariem Souissi ◽  
Julien Pernier ◽  
Olivier Rossier ◽  
Gregory Giannone ◽  
Christophe Le Clainche ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Membrane ◽  
Transmembrane Protein ◽  
Model Membranes ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Good Practices ◽  
Adhesion Proteins ◽  
Controlled Conditions

Giant unilamellar vesicles (GUV) are powerful tools to explore physics and biochemistry of the cell membrane in controlled conditions. For example, GUVs were extensively used to probe cell adhesion, but often using non-physiological linkers, due to the difficulty of incorporating transmembrane adhesion proteins into model membranes. Here we describe a new protocol for making GUVs incorporating the transmembrane protein integrin using gel-assisted swelling. We report an optimised protocol, enumerating the pitfalls encountered and precautions to be taken to maintain the robustness of the protocol. We characterise intermediate steps of small proteoliposome formation and the final formed GUVs. We show that the integrin molecules are successfully incorporated and are functional.

Application of High-Throughput Automated Patch-Clamp Electrophysiology to Study Voltage-Gated Ion Channel Function in Primary Cortical Cultures

2020 ◽  
Vol 25 (5) ◽  
pp. 447-457 ◽  
Author(s):  
May Fern Toh ◽  
Julie M. Brooks ◽  
Tim Strassmaier ◽  
Rodolfo J. Haedo ◽  
Corey B. Puryear ◽  
...  
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Ion Channel ◽  
Cortical Neurons ◽  
Microelectrode Array ◽  
Neuronal Marker ◽  
Voltage Gated ◽  
Channel Function ◽  
Endogenous Expression ◽  
Cortical Cultures

Conventionally, manual patch-clamp electrophysiological approaches are the gold standard for studying ion channel function in neurons. However, these approaches are labor-intensive, yielding low-throughput results, and are therefore not amenable for compound profiling efforts during the early stages of drug discovery. The SyncroPatch 384PE has been successfully implemented for pharmacological experiments in heterologous overexpression systems that may not reproduce the function of voltage-gated ion channels in a native, heterogeneous environment. Here, we describe a protocol allowing the characterization of endogenous voltage-gated potassium (Kv) and sodium (Nav) channel function in developing primary rat cortical cultures, allowing investigations at a significantly improved throughput compared with manual approaches. Key neuronal marker expression and microelectrode array recordings of electrophysiological activity over time correlated well with neuronal maturation. Gene expression data revealed high molecular diversity in Kv and Nav subunit composition throughout development. Voltage-clamp experiments elicited three major current components composed of inward and outward conductances. Further pharmacological experiments confirmed the endogenous expression of functional Kv and Nav channels in primary cortical neurons. The major advantages of this approach compared with conventional manual patch-clamp systems include unprecedented improvements in experimental ease and throughput for ion channel research in primary neurons. These efforts demonstrated feasibility for primary neuronal ion channel investigation with the SyncroPatch, providing the foundation for future studies characterizing biophysical changes in endogenous ion channels in primary systems associated with disease or development.

The voltage-gated channel accessory protein KCNE2: multiple ion channel partners, multiple ways to long QT syndrome

2011 ◽  
Vol 13 ◽  
Author(s):  
Jodene Eldstrom ◽  
David Fedida
Keyword(s):  
Ion Channel ◽  
Long Qt Syndrome ◽  
Transmembrane Protein ◽  
Accessory Protein ◽  
Long Qt ◽  
Clinical Role ◽  
Voltage Gated ◽  
Single Pass ◽  
Gated Channel ◽  
Qt Syndrome

The single-pass transmembrane protein KCNE2 or MIRP1 was once thought to be the missing accessory protein that combined with hERG to fully recapitulate the cardiac repolarising current IKr. As a result of this role, it was an easy next step to associate mutations in KCNE2 to long QT syndrome, in which there is delayed repolarisation of the heart. Since that time however, KCNE2 has been shown to modify the behaviour of several other channels and currents, and its role in the heart and in the aetiology of long QT syndrome has become less clear. In this article, we review the known interactions of the KCNE2 protein and the resulting functional effects, and the effects of mutations in KCNE2 and their clinical role.

scholarly journals Functional Expression of TRPV1 Ion Channel in the Canine Peripheral Blood Mononuclear Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3177
Author(s):  
Joanna K. Bujak ◽  
Daria Kosmala ◽  
Kinga Majchrzak-Kuligowska ◽  
Piotr Bednarczyk
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Functional Expression ◽  
Peripheral Blood Mononuclear ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Blood Mononuclear Cells

TRPV1, known as a capsaicin receptor, is the best-described transient receptor potential (TRP) ion channel. Recently, it was shown to be expressed by non-excitable cells such as lymphocytes. However, the data regarding the functional expression of the TRPV1 channel in the immune cells are often contradictory. In the present study, we performed a phylogenetical analysis of the canine TRP ion channels, we assessed the expression of TRPV1 in the canine peripheral blood mononuclear cells (PBMC) by qPCR and Western blot, and we determined the functionality of TRPV1 by whole-cell patch-clamp recordings and calcium assay. We found high expression of TRPV2, -M2, and -M7 in the canine PBMCs, while expression of TRPV1, -V4 and, -M5 was relatively low. We confirmed that TRPV1 is expressed on the protein level in the PBMC and it localizes in the plasma membrane. The whole-cell patch-clamp recording revealed that capsaicin application caused a significant increase in the current density. Similarly, the results from the calcium assay show a dose-dependent increase in intracellular calcium level in the presence of capsaicin that was partially abolished by capsazepine. Our study confirms the expression of TRPV1 ion channel on both mRNA and protein levels in the canine PBMC and indicates that the ion channel is functional.

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