TRP Channel Reconstitution in Lipid Bilayers

2019 ◽  
pp. 143-166
Author(s):  
Eleonora Zakharian
Keyword(s):  
Lipid Bilayers ◽  
Trp Channel ◽  
Channel Reconstitution

scholarly journals Phosphatidylinositol 3,5-Bisphosphate regulates Ca2+ Transport During Yeast Vacuolar Fusion through Activation of the Ca2+ ATPase Pmc1

2019 ◽  
Author(s):  
Gregory E. Miner ◽  
Katherine D. Sullivan ◽  
Annie Guo ◽  
Matthew L. Starr ◽  
EZ C. Ellis ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Lipid Bilayers ◽  
Dual Role ◽  
Snare Complex ◽  
Trp Channel ◽  
Complex Assembly ◽  
Hyperosmotic Shock ◽  
Vacuole Fusion ◽  
Opposing Effects

ABSTRACTThe transport of Ca2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles during hyperosmotic shock become fragmented through fission events that require Ca2+ efflux of their luminal stores through the TRP channel Yvc1. This requires the production of the lipid PI(3,5)P2 by Fab1. Ca2+ is also released during vacuole fusion upon trans-SNARE complex assembly, however, the role of PI(3,5)P2 remains unclear. Here we demonstrate that elevated PI(3,5)P2 levels abolish Ca2+ efflux during fusion, indicating that PI(3,5)P2 has opposing effects on Ca2+ transport in fission versus fusion. Notably, Ca2+ efflux was enhanced when PI(3,5)P2 levels were reduced. Importantly, the effect of PI(3,5)P2 on Ca2+ flux was independent of Yvc1. Rather, the effect was dependent on the Ca2+ pump Pmc1. Vacuoles lacking Pmc1 were resistant to the effects of PI(3,5)P2, while those lacking Yvc1 remained sensitive. Furthermore altering PI(3,5)P2 levels affects the interactions of Pmc1 with the Vo component Vph1 and the R-SNARE Nyv1. We now propose a model in which elevated PI(3,5)P2 activates continued Pmc1 function to prevent the accumulation of released extraluminal Ca2+.SummaryDuring osmotic stress PI(3,5)P2 triggers Ca2+ release from vacuoles. Here we show PI(3,5)P2 stimulates Ca2+ uptake by vacuoles during fusion, illustrating that it has a dual role in Ca2+ transport.

Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 608-609
Author(s):  
Neng-Bo He ◽  
S.W. Hui
Keyword(s):  
Lipid Bilayers ◽  
Erythrocyte Membrane ◽  
Lipid Membranes ◽  
Surface Film ◽  
Biological Membranes ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
Black Lipid Membranes ◽  
Fine Mesh ◽  
Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

Effect of Integral Proteins on Frozen Membrane Fracture

1980 ◽  
Vol 38 ◽  
pp. 756-759 ◽  
Author(s):  
S. Kirchanski ◽  
D. Branton
Keyword(s):  
Lipid Bilayers ◽  
Freeze Fracture ◽  
Covalent Bond ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Experimental Protocol ◽  
Transmembrane Glycoprotein ◽  
Bond Breakage ◽  
Human Erythrocyte Membranes ◽  
Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

Late Stages of the “Pearling" Instability in Lipid Bilayers

1997 ◽  
Vol 7 (9) ◽  
pp. 1185-1204 ◽  
Author(s):  
J. L. Coveas ◽  
S. T. Milner ◽  
W. B. Russel
Keyword(s):  
Lipid Bilayers ◽  
Late Stages

GLP-1 based multi-agonists-induced signaling includes profound TRP channel involvement in insulin secretion

2017 ◽  
Author(s):  
Noushafarin Khajavi ◽  
Brian Finan ◽  
Oliver Kluth ◽  
Stefan Mergler ◽  
Timo Muller ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Trp Channel

Improved micro-impedance spectroscopy to determine cell barrier properties

2020 ◽  
Vol 4 (3) ◽  
pp. 150-155 ◽  
Author(s):  
Md. Mehadi Hasan Sohag ◽  
Olivier Nicoud ◽  
Racha Amine ◽  
Abir Khalil-Mgharbel ◽  
Jean-Pierre Alcaraz ◽  
...  
Keyword(s):  
Impedance Spectroscopy ◽  
Epithelial Cells ◽  
Lipid Bilayers ◽  
Cultured Cells ◽  
Cell Model ◽  
Measurement Techniques ◽  
Cell Monolayer ◽  
Microfluidic Channel ◽  
Small Surface ◽  
Resistance Pathway

AbstractThe goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm2) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.

Direct Measurement of Charge Reversal on Lipid Bilayers using Heterodyne-Detected Second Harmonic Generation Spectroscopy

2019 ◽  
Author(s):  
HanByul Chang ◽  
Paul Ohno ◽  
Yangdongling Liu ◽  
Franz Geiger
Keyword(s):  
Lipid Bilayers ◽  
Surface Potential ◽  
Second Harmonic ◽  
Fluid Phase ◽  
Cationic Polyelectrolyte ◽  
Charge Reversal ◽  
Buried Interfaces ◽  
Phase Transition Temperatures ◽  
Complementary Techniques ◽  
Order Susceptibility

We report the detection of charge reversal induced by the adsorption of a cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), to buried supported lipid bilayers (SLBs), used as idealized model biological membranes. We observe changes in the surface potential in isolation from other contributors to the total SHG response by extracting the phase-shifted potential-dependent third-order susceptibility from the overall SHG signal. We demonstrate the utility of this technique in detecting both the sign of the surface potential and the point of charge reversal at buried interfaces without any prior information or complementary techniques<i>.</i>Furthermore, isolation of the second-order susceptibility contribution from the overall SHG response allows us to directly monitor changes in the Stern Layer. Finally, we characterize the Stern and Diffuse Layers over single-component SLBs formed from three different zwitterionic lipids of different gel-to-fluid phase transition temperatures (T<sub>m</sub>s). We determine whether the surface potential changes with the physical phase state (gel, transitioning, or fluid) of the SLB and incorporate 20 percent of negatively charged lipids to the zwitterionic SLB to investigate how the surface potential changes with surface charge.

Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

2018 ◽  
Author(s):  
Luke Jordan ◽  
Nathan Wittenberg
Keyword(s):  
Lipid Bilayers ◽  
Binding Kinetics ◽  
Supported Lipid Bilayers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Supported Lipid Bilayer ◽  
Head Groups ◽  
Lipid Diffusion ◽  
Kinetics Of ◽  
Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

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