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

2018 ◽  
Vol 115 (8) ◽  
pp. E1789-E1798 ◽  
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.

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

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

Mechanically Stable Lipid Bilayers in Teflon-Coated Silicon Chips for Single-Channel Recordings

2012 ◽  
Vol 4 (1) ◽  
pp. 2-7 ◽  
Author(s):  
Azusa Oshima ◽  
Ayumi Hirano-Iwata ◽  
Tomohiro Nasu ◽  
Yasuo Kimura ◽  
Michio Niwano
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Single Channel Recordings ◽  
Silicon Chips

Ryanodine receptor dysfunction in porcine stunned myocardium

1997 ◽  
Vol 273 (2) ◽  
pp. H796-H804 ◽  
Author(s):  
C. Valdivia ◽  
J. O. Hegge ◽  
R. D. Lasley ◽  
H. H. Valdivia ◽  
R. Mentzer
Keyword(s):  
Coronary Artery ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Myocardial Stunning ◽  
Single Channel ◽  
Stunned Myocardium ◽  
Wall Thickening ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Single Channel Recordings

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.

Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor

2000 ◽  
Vol 351 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Salim M. HAYEK ◽  
Xinsheng ZHU ◽  
Manjunatha B. BHAT ◽  
Jiying ZHAO ◽  
Hiroshi TAKESHIMA ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Expression System ◽  
Binding Assays ◽  
Release Channel ◽  
Important Region ◽  
Functional Consequences ◽  
Increased Sensitivity ◽  
Regulatory Functions

A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872–1923, is involved in Ca 2+-dependent regulation of the Ca2+-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca2+ exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca2+ concentrations respectively. To characterize the role of D3 in the Ca2+-dependent regulation of the Ca2+-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (∆D3-RyR1) using a heterologous expression system, [3H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca2+-dependent regulation of RyR1, but did not alter [3H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca2+-dependence curve of ∆D3-RyR1 is broader, reflecting increased sensitivity to Ca2+ activation and decreased sensitivity to Ca2+ inactivation. In addition, ∆D3-RyR1 was more resistant to inhibition by Mg2+. Comparison of the effect of caffeine on wt-RyR1 and ∆D3-RyR1 suggested that D3 is an important region of RyR that participates in Ca2+-dependent activation and inactivation of the Ca2+-release channel.

Ethanol enhances caffeine-induced Ca2+-release channel activation in skeletal muscle sarcoplasmic reticulum

1997 ◽  
Vol 272 (2) ◽  
pp. C622-C627 ◽  
Author(s):  
T. Oba ◽  
M. Koshita ◽  
M. Yamaguchi
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Skeletal Muscles ◽  
Single Channel ◽  
Channel Activity ◽  
Electrical Parameters ◽  
Open Probability ◽  
Release Channel ◽  
Channel Current ◽  
Open Time

When sarcoplasmic reticulum (SR) vesicles prepared from frog skeletal muscles were actively loaded with Ca2+, pretreatment of the SR with 2.2 mM (0.01%) ethanol for 30 s significantly potentiated 5 mM caffeine-induced release of Ca2+ from 16.7 +/- 3.7 nmol/mg protein in control without ethanol to 28.0 +/- 2.6 nmol/mg (P < 0.05, n = 5). Ethanol alone caused no release of Ca2+ from the SR. Exposure of the Ca2+-release channel, incorporated into planar lipid bilayers, to 2 mM caffeine significantly increased open probability (Po) and mean open time, but unitary conductance was not affected. Ethanol (2.2 mM) enhanced caffeine-induced Ca2+-release channel activity, with Po reaching 3.02-fold and mean open time 2.85-fold the values in the absence of ethanol. However, ethanol alone did not affect electrical parameters of single-channel current, over a concentration range of 2.2 mM (0.01%) to 217 mM (1%). The synergistic action of ethanol and caffeine on the channel activity could be attributable to enhancement of caffeine-induced release of Ca2+ from the SR vesicles in the presence of ethanol.

scholarly journals Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum. Activation by Ca2+ and ATP and modulation by Mg2+.

1986 ◽  
Vol 88 (5) ◽  
pp. 573-588 ◽  
Author(s):  
J S Smith ◽  
R Coronado ◽  
G Meissner
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Calcium Release ◽  
Single Channel ◽  
Ca Channel ◽  
Channel Measurements ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Release Channel

A high-conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy-density skeletal muscle sarcoplasmic reticulum (SR) fractions into planar lipid bilayers of the Mueller-Rudin type. cis Ca2+ in the range of 2-950 microM increased open probability (Po) in single channel records without affecting open event lifetimes. Millimolar ATP was found to be as good as or better than Ca2+ in activation; however, both Ca2+ and ATP were required to fully activate the channel, i.e., to bring Po = 1. Exponential fits to open and closed single channel lifetimes suggested that the channel may exist in many distinct states. Two open and two closed states were identified when the channel was activated by either Ca2+ or ATP alone or by Ca2+ plus nucleotide. Mg2+ was found to permeate the SR Ca channel in a trans-to-cis direction such that iMg2+/iCa2+ = 0.40. cis Mg2+ was inhibitory and in single channel recordings produced an unresolvable flickering of Ca- and nucleotide-activated channels. At nanomolar cis Ca2+, 4 microM Mg2+ completely inhibited nucleotide-activated channels. In the presence of 2 microM cis Ca2+, the nucleotide-activated macroscopic Ba conductance was inhibited by cis Mg2+ with an IC50 equal to 1.5 mM.

scholarly journals Bitstream Photon Counting Chirped AM Lidar with Digital I/Q Demodulation

2020 ◽  
Author(s):  
Brian Redman
Keyword(s):  
Integrated Circuits ◽  
Single Channel ◽  
Signal To Noise Ratio ◽  
Photon Counting ◽  
Complementary Metal Oxide Semiconductor ◽  
Local Oscillator ◽  
Oxide Semiconductor ◽  
Signal To Noise ◽  
Receiver Architecture ◽  
Noise Ratio

This paper is a follow-up to two previous papers, one introducing the new bitstream Photon Counting Chirped Amplitude Modulation (AM) Lidar (PC-CAML) with the unipolar Digital Logic Local Oscillator (DLLO) concept, and the other paper introducing the improvement thereof using the bipolar DLLO. In that previous work, there was only a single channel of digital mixing of the DLLO with the received photon counting signal. This paper introduces a new bitstream PC-CAML receiver architecture with an in-phase (I) digital mixing channel and a quadrature phase (Q) digital mixing channel for digital I/Q demodulation with the bipolar DLLO to improve the signal-to-noise ratio (SNR) by 3 dB compared to that for the single digital mixing channel with the bipolar DLLO and by 5.5 dB compared to that for the single digital mixing channel with the unipolar DLLO. (patent pending) The bipolar DLLO with digital I/Q demodulation architecture discussed in this paper retains the key advantages of the previous bitstream PC-CAML with a DLLO systems since it also replaces bulky, power-hungry, and expensive wideband RF analog electronics with digital components that can be implemented in inexpensive silicon complementary metal-oxide-semiconductor (CMOS) read-out integrated circuits (ROICs) to make the bitstream PC-CAML with a DLLO more suitable for compact lidar-on-a-chip systems and lidar array receivers than previous PC-CAML systems. This paper introduces the bipolar DLLO with digital I/Q demodulation receiver architecture for bitstream PC-CAML and presents the initial signal-to-noise ratio (SNR) theory with comparisons to Monte Carlo simulation results.

scholarly journals Antibodies as probes for ligand gating of single sarcoplasmic reticulum Ca2+-release channels

1991 ◽  
Vol 273 (2) ◽  
pp. 449-457 ◽  
Author(s):  
M Fill ◽  
R Mejia-Alvarez ◽  
F Zorzato ◽  
P Volpe ◽  
E Stefani
Keyword(s):  
Amino Acid ◽  
Lipid Bilayers ◽  
Binding Sites ◽  
Single Channel ◽  
Channel Gating ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Release Channel ◽  
Primary Amino Acid Sequence ◽  
Primary Amino

A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody-induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti-RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand-binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single-channel level.

Single cardiac sarcoplasmic reticulum Ca2+-release channel: activation by caffeine

1989 ◽  
Vol 256 (2) ◽  
pp. H328-H333 ◽  
Author(s):  
E. Rousseau ◽  
G. Meissner
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Ruthenium Red ◽  
Planar Lipid Bilayers ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Channel Activation ◽  
Contraction Coupling

Caffeine is thought to affect excitation-contraction coupling in cardiac muscle by activating the sarcoplasmic reticulum (SR) Ca2+-release channel. The effect of caffeine at the single channel level was studied by incorporating canine cardiac SR vesicles into planar lipid bilayers. Cardiac Ca2+-release channels were activated in a steady-state manner by millimolar cis-caffeine and displayed a unitary conductance (77 pS in 50 mM Ca2+ trans) similar to that previously observed for the Ca2+-activated cardiac channel. The caffeine-activated channel was moderately sensitive to the voltage applied across the bilayer, was sensitive to further activation by ATP, and was inhibited by Mg2+ and ruthenium red. Kinetic analysis showed that at low Ca2+ concentration, caffeine activated the channel by increasing the frequency and the duration of open events.

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