Single-Molecule Patch-Clamp FRET Anisotropy Microscopy Studies of NMDA Receptor Ion Channel Activation and Deactivation under Agonist Ligand Binding in Living Cells

AbstractUnderstanding and accurately quantifying ion channel molecule gating in real time is vital for knowledge of cell membrane behaviour, drug discovery and toxicity screening. Doing this with single-molecule resolution first requires the detection of individual protein pore opening and closing transitions and construction of a so-called idealised record which indicates sample-point by samplepoint whether a given molecule is open or closed. Creating this can be difficult, since patch-clamp electrophysiology data can be noisy or contain multiple ion channel molecules. We have recently developed a deep learning model to achieve this called Deep-Channel, but further development is limited by the massive datasets need to train and validate models. In the past, this problem has been tackled by simulation of single molecule activity from Markov models with the addition of pseudo-random noise. In the present report we develop a new method to synthesise raw data, based on generative adversarial networks (GANs). The limitation to direct application of a GAN with this method has been that whilst there are methods to generate classified output image by image, there has been no method to generate an entire timeseries with parallel idealisation, sample-point by sample-point. In this paper, we over-come this problem with DeepGANnel, a model that splits training data raw and parallel idealised data into different rows of image windows and passes these data through a progressive-GAN. This new methodology allows generation of realistic, idealisation synchronised single molecule patch-clamp data, without the biases inherent in pseudorandom simulation methods. This method will be useful for development of single molecule analysis methods and may in the future prove useful for generation of biological models including single molecule resolution stochastic data. The model is easily extendable to other timeseries data requiring parallel labelling, such as labelled ECG.

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Probing ion channel conformational dynamics using simultaneous single-molecule ultrafast spectroscopy and patch-clamp electric recording

Applied Physics Letters ◽

10.1063/1.1652228 ◽

2004 ◽

Vol 84 (10) ◽

pp. 1792-1794 ◽

Cited By ~ 20

Author(s):

Greg Harms ◽

Galya Orr ◽

H. Peter Lu

Keyword(s):

Patch Clamp ◽

Ion Channel ◽

Single Molecule ◽

Ultrafast Spectroscopy ◽

Conformational Dynamics

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Effect of the novel high-affinity glycine-site N-methyl-d-aspartate antagonist ACEA-1021 on 125I-MK-801 binding after subdural hematoma in the rat: an in vivo autoradiographic study

Journal of Neurosurgery ◽

10.3171/jns.1996.85.4.0655 ◽

1996 ◽

Vol 85 (4) ◽

pp. 655-661 ◽

Cited By ~ 25

Author(s):

Xiao Di ◽

Ross Bullock

Keyword(s):

Nmda Receptor ◽

Ion Channel ◽

Subdural Hematoma ◽

Nmda Antagonist ◽

Channel Activation ◽

Content Type ◽

Mk 801 ◽

High Affinity ◽

Fine Print

✓ Acute subdural hematoma (SDH) complicates 20% of severe human head injuries and causes death or severe disability in 60% of these cases, due to brain swelling and high intracranial pressure. Although the mechanisms for these phenomena are unknown, previous studies have implicated excitatory amino acid—mediated mechanisms in both humans and animal models. The authors therefore performed in vivo autoradiography using 125I-MK-801, a high-affinity noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, as a tracer to evaluate NMDA ion channel activation spatially and temporally as a factor causing cytotoxic swelling. Acute SDH was induced in 16 anesthetized rats using 0.4 ml autologous venous blood. Fifty microcuries of 125I-MK-801 was injected via an aortic arch cannula 30 minutes after onset of SDH. The effect of a new putatively neuroprotective drug, ACEA-1021, a glycine-specific binding site NMDA antagonist, on 125I-MK-801 binding was tested on five animals. “Nonspecific” 125I-MK-801 binding in the rat brain was assessed by pretreatment with “cold” (nonradiolabeled) MK-801 in five more animals. Four hours later the animals were sacrificed and brain sections were apposed to radiation-detecting high-sensitivity photographic film with precalibrated plastic standards for 4 weeks. A striking and highly significant 1.7- to 4.8-fold increase in 125I-MK-801 binding was seen in the penumbra of viable tissue surrounding the ischemic zone beneath the acute SDH, when compared to contralateral hemisphere binding (p < 0.001). The MK-801 pretreatment markedly reduced 125I-MK-801 uptake in this penumbral zone (4.73 ± 0.36 nCi/mg control vs. 2.85 ± 0.08 nCi/mg cold MK-801; p < 0.0001), indicating that the increased binding in the penumbra of the lesion was due to NMDA ion channel activation. Pretreatment with ACEA-1021 reduced 125I-MK-801 uptake by 28% (3.41 ± 0.26 nCi/mg vs. 4.73 ± 0.36 nCi/mg; p < 0.05), indicating that this agent prevents opening of the NMDA ion channel and, thus, exposure of its receptor for MK-801 binding. These studies show intense foci of penumbral NMDA receptor-mediated ion channel activation after onset of SDH, which is markedly reduced by an NMDA antagonist. Such agents are thus likely to reduce cell swelling after SDH occurs.

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