Single channel recordings of α-hemolysin reconstituted in S-layer-supported lipid bilayers

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

Download Full-text

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712313115 ◽

2018 ◽

Vol 115 (8) ◽

pp. E1789-E1798 ◽

Cited By ~ 7

Author(s):

Andreas J. W. Hartel ◽

Peijie Ong ◽

Indra Schroeder ◽

M. Hunter Giese ◽

Siddharth Shekar ◽

...

Keyword(s):

Integrated Circuits ◽

Lipid Bilayers ◽

Temporal Resolution ◽

Single Channel ◽

Low Noise ◽

Oxide Semiconductor ◽

Distribution Analysis ◽

Release Channel ◽

Single Channel Recordings ◽

High Bandwidth

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.

Download Full-text

Immobilizing Channel Molecules in Artificial Lipid Bilayers for Simultaneous Electrical and Optical Single Channel Recordings

Cell Signaling Reactions ◽

10.1007/978-90-481-9864-1_5 ◽

2010 ◽

pp. 107-120

Author(s):

Toru Ide ◽

Minako Hirano ◽

Takehiko Ichikawa

Keyword(s):

Lipid Bilayers ◽

Single Channel ◽

Single Channel Recordings ◽

Artificial Lipid Bilayers

Download Full-text

Incorporation of RyR2 and Other Ion Channels into Nanopore Based Planar Lipid Bilayers for Low Noise Single Channel Recordings

Biophysical Journal ◽

10.1016/j.bpj.2009.12.2928 ◽

2010 ◽

Vol 98 (3) ◽

pp. 539a-540a

Author(s):

Prithwish Pal ◽

Geoffrey A. Barrall ◽

Ariel L. Escobar ◽

Melissa A. Poquette ◽

Patricio Velez ◽

...

Keyword(s):

Ion Channels ◽

Lipid Bilayers ◽

Single Channel ◽

Low Noise ◽

Planar Lipid Bilayers ◽

Single Channel Recordings

Download Full-text

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

10.26434/chemrxiv.7505330 ◽

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.

Download Full-text