scholarly journals Gating Transitions in Bacterial Ion Channels Measured at 3 μs Resolution

2004 ◽  
Vol 124 (2) ◽  
pp. 151-161 ◽  
Author(s):  
George Shapovalov ◽  
Henry A. Lester
Keyword(s):  
Ion Channels ◽  
Single Molecule ◽  
Time Course ◽  
Single Channel ◽  
Low Noise ◽  
State Transitions ◽  
Channel Measurements ◽  
Clear Trend ◽  
Single Molecule Level ◽  
High Conductance

Ion channels of high conductance (>200 pS) are widespread among prokaryotes and eukaryotes. Two examples, the Escherichia coli mechanosensitive ion channels Ec-MscS and Ec-MscL, pass currents of 125–300 pA. To resolve temporal details of conductance transitions, a patch-clamp setup was optimized for low-noise recordings at a time resolution of 3 μs (10–20 times faster than usual). Analyses of the high-resolution recordings confirm that Ec-MscL visits many subconductance states and show that most of the intersubstate transitions occur more slowly than the effective resolution of 3 μs. There is a clear trend toward longer transition times for the larger transitions. In Ec-MscS recordings, the majority of the observed full conductance transitions are also composite. We detected a short-lived (∼20 μs) Ec-MscS substate at 2/3 of full conductance; transitions between 2/3 and full conductance did not show fine structure and had a time course limited by the achieved resolution. Opening and closing transitions in MscS are symmetrical and are not preceded or followed by smaller, rapid currents (“anticipations” or “regrets”). Compared with other, lower-conductance channels, these measurements may detect unusually early states in the transitions from fully closed to fully open. Increased temporal resolution at the single-molecule level reveals that some elementary steps of structural transitions are composite and follow several alternative pathways, while others still escape resolution. High-bandwidth, low-noise single-channel measurements may provide details about state transitions in other high-conductance channels; and similar procedures may also be applied to channel- and nanopore-based single-molecule DNA measurements.

scholarly journals Mechanism of modulation of AMPA receptors by TARP-γ8

2019 ◽  
Vol 152 (1) ◽  
Author(s):  
Elisa Carrillo ◽  
Sana A. Shaikh ◽  
Vladimir Berka ◽  
Ryan J. Durham ◽  
Douglas B. Litwin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ampa Receptors ◽  
Single Channel ◽  
Regulatory Proteins ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
High Conductance State ◽  
Tightly Coupled ◽  
And Function ◽  
High Conductance

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate-activated α-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) receptors. In neurons, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs). Assembly with TARP γ8 alters the biophysical properties of the receptor, producing resensitization currents in the continued presence of glutamate. Using single-channel recordings, we show that under resensitizing conditions, GluA2 AMPA receptors primarily transition to higher conductance levels, similar to activation of the receptors in the presence of cyclothiazide, which stabilizes the open state. To study the conformation associated with these states, we have used single-molecule FRET and show that this high-conductance state exhibits tighter coupling between subunits in the extracellular parts of the receptor. Furthermore, the dwell times for the transition from the tightly coupled state to the decoupled states correlate to longer open durations of the channels, thus correlating conformation and function at the single-molecule level.

Permeation of Antibiotics through Escherichia coli OmpF and OmpC Porins

2010 ◽  
Vol 15 (3) ◽  
pp. 302-307 ◽  
Author(s):  
Kozhinjampara R. Mahendran ◽  
Mohamed Kreir ◽  
Helge Weingart ◽  
Niels Fertig ◽  
Mathias Winterhalter
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Single Molecule Level ◽  
Solution Exchange ◽  
Automated Patch Clamp ◽  
Low Capacitance ◽  
Bacterial Porins

A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo–giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics—cephalosporins and fluoroquinolones—through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level.

scholarly journals Monitoring HIV-1 Assembly in Living Cells: Insight from Dynamic and Single Molecule Microscopy

2018 ◽  
Author(s):  
Kaushik Inamdar ◽  
Charlotte Floderer ◽  
Cyril Favard ◽  
Delphine Muriaux
Keyword(s):  
Host Cell ◽  
Single Molecule ◽  
High Speed ◽  
Time Course ◽  
Super Resolution ◽  
Living Cells ◽  
Single Molecule Level ◽  
Host Cell Factors ◽  
Immature Virus ◽  
Hiv 1

HIV-1 assembly is a complex mechanism taking place at the plasma membrane of the host cell. It requires nice spatial and temporal coordination to end up with a full immature virus. Researchers have extensively studied HIV-1 assembly molecular mechanism during the past decades, in order to dissect the respective roles of viral proteins, viral genome and host cell factors. Nevertheless, the time course of the process has been observed in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resolution now permit to study assemblies and their consequences at the single molecule level within (living) cells. In this review, after a short description of these new approaches, we will show how HIV-1 assembly in cells has been revisited using these advanced super resolution microscopy techniques and how much it could make a bridge in studying assembly from the single molecule to the host cell.

scholarly journals Active DNA unwinding and transport by a membrane-adapted helicase nanopore

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ke Sun ◽  
Changjian Zhao ◽  
Xiaojun Zeng ◽  
Yuejia Chen ◽  
Xin Jiang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Single Cell Analysis ◽  
Single Channel ◽  
Vital Role ◽  
Live Cell ◽  
Bovine Papillomavirus ◽  
Nanoscale Transport ◽  
Single Molecule Level

Abstract Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

scholarly journals Gating currents associated with Na channels in canine cardiac Purkinje cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 439-457 ◽  
Author(s):  
D A Hanck ◽  
M F Sheets ◽  
H A Fozzard
Keyword(s):  
Purkinje Cells ◽  
Single Channel ◽  
State Transitions ◽  
Na Channel ◽  
Channel Measurements ◽  
Na Channels ◽  
Gating Currents ◽  
Closed State ◽  
Conductance Curve ◽  
Cardiac Purkinje Cells

Gating currents (Ig) were recorded in single canine cardiac Purkinje cells at 10-12 degrees C. Ig characteristics corresponded closely to macroscopic INa characteristics and appeared to exhibit little contamination from other voltage-gated channels. Charge density predicted by peak INa was 0.14-0.22 fC micron -2 and this compared well with the measured value of 0.19 +/- 0.10 fC micron -2 (SD; n = 28). The charge-voltage relationship rose over a voltage similar to the peak INa conductance curve. The midpoints of the two relationships were not significantly different although the conductance curve was 1.5 +/- 0.3 (SD; n = 9) times steeper. Consistent with this observation, which predicted that a large amount of the gating charge would be associated with transitions close to the open state, an analysis of activation from Hodgkin-Huxley fits to the macroscopic currents showed that tau m corresponded well with a prominent component of Ig. Ig relaxations fitted two exponentials better than one over the range of voltages in which Na channels were activated. When the holding potential was hyperpolarized, relaxation of Ig during step depolarizations to 0 mV was prolonged but there was no substantial increase in charge, further suggesting that early closed-state transitions are less in charge, further suggesting that early closed-state transitions are less voltage dependent. The single cardiac Purkinje cell appears to be a good candidate for combining Ig and single-channel measurements to obtain a kinetic description of the cardiac Na channel.

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
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