scholarly journals Preparation of Artificial Bilayers for Electrophysiology Experiments

10.3791/1033 ◽  
2008 ◽  
Author(s):  
Ruchi Kapoor ◽  
Jung H. Kim ◽  
Helgi Ingolfson ◽  
Olaf Sparre Andersen
Keyword(s):  
Artificial Bilayers

scholarly journals Examination of the Interaction between a Membrane Active Peptide and Artificial Bilayers by Dual Polarisation Interferometry

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Jennifer Payne ◽  
Tzong-Hsien Lee ◽  
Marilyn Anderson ◽  
Marie-Isabel Aguilar
Keyword(s):  
Active Peptide ◽  
Dual Polarisation Interferometry ◽  
Artificial Bilayers

Comparison of large-conductance Ca(2+)-activated K+ channels in artificial bilayer and patch-clamp experiments

1994 ◽  
Vol 266 (3) ◽  
pp. C601-C610 ◽  
Author(s):  
C. L. Kapicka ◽  
A. Carl ◽  
M. L. Hall ◽  
A. L. Percival ◽  
B. W. Frey ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Apparent Distance ◽  
Bk Channels ◽  
Open Probability ◽  
K Channels ◽  
Artificial Lipid Bilayers ◽  
Excised Patches ◽  
Lipid Environment ◽  
Artificial Bilayers ◽  
Channel Properties

We compared the gating, ion conduction, and pharmacology of large-conductance Ca(2+)-activated K+ channels (BK channels) from canine colon in artificial lipid bilayers and in excised patches. Both protocols identified 270-pS K(+)-selective channels activated by depolarization and Ca2+ (approximately 130-mV shift of half-activation voltage per 10-fold change in Ca2+) that were inhibited by extracellular tetraethylammonium (TEA) and charybdotoxin. These similarities suggest that the same BK channels are studied in the two techniques. However, we found three quantitative differences between channels in artificial bilayers and patches. 1) Channels in artificial bilayers required fivefold higher free Ca2+ or 80-mV stronger depolarization for activation. 2) The voltage dependence of TEA block was smaller for channels in artificial bilayers. The apparent distance across the membrane field for the TEA binding site was 0.031 for channels in artificial bilayers and 0.23 for channels in patches. 3) ATP (2 mM) decreased open probability (Po) of channels in artificial bilayers, whereas channels in patches were unaffected. Neither GTP nor UTP reduced Po of channels in artificial bilayers. It is possible that these differences may be due to a lack of molecular identity between the channels studied in the two protocols. Alternatively, they may be attributed to alterations in channel properties during reconstitution or to influences of the artificial lipid environment.

Properties of ionic transport through phospholipid-glycolipid artificial bilayers

1982 ◽  
Vol 693 (1) ◽  
pp. 165-172 ◽  
Author(s):  
Franco Gambale ◽  
Mauro Robello ◽  
Cesare Usai ◽  
Carla Marchetti
Keyword(s):  
Ionic Transport ◽  
Artificial Bilayers

AMPA and kainate-operated channels reconstituted in artificial bilayers

FEBS Letters ◽  
1991 ◽  
Vol 281 (1-2) ◽  
pp. 27-29 ◽  
Author(s):  
A. Ambrosini ◽  
E.A. Barnard ◽  
G. Prestipino
Keyword(s):  
Artificial Bilayers

scholarly journals Can Lipids be used as Mobility Standards in Artificial Bilayers?

2015 ◽  
Vol 108 (2) ◽  
pp. 181a
Author(s):  
Wladimir Urbach ◽  
Vladimir Adrien ◽  
Gamal Rayan ◽  
Nicolas Taulier ◽  
Patrick Fuchs
Keyword(s):  
Artificial Bilayers

The Power of Freeze-Fracture Technique in Electron Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 1212-1213
Author(s):  
B. Papahadjopoulos-Sternberg
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayers ◽  
High Vacuum ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Model Systems ◽  
Transmission Electron ◽  
A New Technique ◽  
Freeze Fracture Electron Microscopy ◽  
Artificial Bilayers

In the early 1960s, concerns about artifacts in preparing biological material for electron microscopy led to a new technique whereby samples are rapidly frozen, fractured under high vacuum, the fractured surfaces shadowed and replicated with a thin metal-carbon coat, and the cleaned replica examined in a transmission electron microscope. Pioneered by Moor and Muhlethaler subcellular structures are revealed with extraordinary three-dimensional clarity at near-molecular resolution. Furthermore, it was observed and proven at model systems as well as biological membranes that lipid bilayers split along their hydrophobic interior during freeze-fracture procedure. Therefore, freeze-fracture electron microscopy (FFEM) has the unique advantage of accessing the hydrophobic interior of biological (FIG.l, 5 and 6) as well as artificial bilayers (FIG. 2, 4, 5, and 6). Here it permits study of pattern generated by intrinsic proteins as well as lipids (FIG. 1 and 2).

scholarly journals The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level

1998 ◽  
Vol 330 (1) ◽  
pp. 559-564 ◽  
Author(s):  
C. Edwin THROWER ◽  
J. A. Edward LEA ◽  
P. Alan DAWSON
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Artificial Bilayers

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

The influence of sterols on the conformation of recombinant mitochondrial porin in detergent

2008 ◽  
Vol 86 (6) ◽  
pp. 539-545 ◽  
Author(s):  
Denice C. Bay ◽  
Joe D. O’Neil ◽  
Deborah A. Court
Keyword(s):  
Amino Acids ◽  
High Resolution ◽  
Secondary Structure ◽  
Sodium Dodecylsulfate ◽  
Aromatic Amino Acids ◽  
Structural Studies ◽  
High Concentration ◽  
Mitochondrial Porin ◽  
Voltage Dependent ◽  
Artificial Bilayers

Mitochondrial porins (voltage-dependent anion-selective channels, VDAC) are key contributors to cellular metabolism. When isolated from mitochondria porins copurify with sterols, and some isolated forms of the protein require sterol for insertion into artificial membranes. Nonetheless, the contributions of sterols to the folded state of mitochondrial porin are not understood. Recently, with the goal of high-resolution structural studies, several laboratories have developed methods for folding recombinant porins at high concentration in detergent. In the present study, recombinant Neurospora crassa porin solubilized in detergent–sterol mixtures was examined. Sterols do not significantly alter the secondary structure of porin in lauryl dimethylamine oxide, nor in a mixture of sodium dodecylsulfate and dodecylmaltopyranoside. However, as detected by near-UV circular dichroism spectropolarimetry and fluorescence spectroscopy, the environments surrounding the aromatic amino acids in the detergent–sterol solubilized protein are measurably different from those in detergent alone. Furthermore, the effects are different in the presence of ergosterol, the native sterol in fungal mitochondria, and cholesterol. While these influences on the tertiary arrangement of detergent-solubilized porin are subtle, they may contribute to the generation of a form of the protein competent for insertion into the artificial bilayers used for electrophysiological analyses, and should be considered in future structural studies of porin.

Translocation of the cell-penetrating Tat peptide across artificial bilayers and into living cells.

2005 ◽  
Vol 72 ◽  
pp. 199-209 ◽  
Author(s):  
Paul Curnow ◽  
Harry Mellor ◽  
David J. Stephens ◽  
Mark Lorch ◽  
Paula J. Booth
Keyword(s):  
Catalytic Domain ◽  
Cysteine Residue ◽  
Live Cells ◽  
Tat Protein ◽  
Tat Peptide ◽  
Fluorescence Confocal Microscopy ◽  
Activating Transcription Factor ◽  
Artificial Bilayers ◽  
Hiv 1

The ability of a short, charged peptide to penetrate synthetic DOPC (1,2-dioleoyl-sn-3-glycerophosphocholine) liposomes was investigated by fluorescence confocal microscopy. The peptide, termed Tat (trans-activating transcription factor), was a 14-mer derived from the region of the HIV-1 Tat protein responsible for cellular internalization. This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-iodoacetamidofluorescein) or A568 (Alexa Fluor 568). The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx. 258C) in the absence of pH gradient, ATP or other energy source. The uptake of the Tat-A568 conjugate in unfixed, live HeLa cells was found to be via endocytosis, as expected. In contrast, when the peptide was attached to an IAF-labelled 25 kDa protein corresponding to the catalytic domain of Clostridium botulinum C3 exotoxin, this larger, Tat-C3-IAF construct was not able to enter liposomes, although it localized similarly to Tat-A568 in live cells. The data suggest that Tat peptide can cross synthetic bilayers spontaneously in vitro, but that size and type of cargo may limit this behaviour.

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