Isolation of Living Neurons from Human Elderly Brains Using the Immunomagnetic Sorting DNA-Linker System

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

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New approaches to the design of analgesic medicinal substances

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2021-0286 ◽

2021 ◽

Author(s):

Ilya V. Rogachevskii ◽

Vera B. Plakhova ◽

Valentina A. Penniyaynen ◽

Stanislav G. Terekhin ◽

Svetlana A. Podzorova ◽

...

Keyword(s):

Functional Groups ◽

Receptor Binding ◽

Therapeutic Potential ◽

Medicinal Substance ◽

Neuron Membrane ◽

Comenic Acid ◽

Structural Criterion ◽

Calcium Cations ◽

Medicinal Substances ◽

Living Neurons

A gamma-pyrone derivative, comenic acid, activates the opioid-like receptor-mediated signaling pathway that modulates the NaV1.8 channels in the primary sensory neuron membrane. These channels are responsible for generation of the nociceptive signal; gamma-pyrones can therefore have a great therapeutic potential as analgesics, and this effect deserves a deeper understanding. The novelty of our approach to the design of a medicinal substance is based on a combination of the data obtained on living neurons using very sensitive physiological methods and the results of quantum-chemical calculations. This approach allows to correlate the molecular structure of gamma-pyrones with their ability to evoke a physiological response of the neuron. Comenic acid can bind two calcium cations. One of them is chelated by the carbonyl and the hydroxyl functional groups, while another one forms the salt bond with the carboxylate anion. Calcium-bound gamma-pyrones are fundamentally different in electrostatic properties from the free gamma-pyrone molecules. These two calcium ions are the key elements involved in ligand-receptor binding. It is very likely ion-ionic interactions between these cations and anionic functional groups of the opioid-like receptor that activate the latter. The calculated intercationic distance of 9.5 Å is a structural criterion for effective ligand-receptor binding of calcium-bound gamma-pyrones.

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Imaging Ion Channel Dynamics in Living Neurons by Fluorescence Microscopy

Methods in Neurosciences - Ion Channels of Excitable Cells ◽

10.1016/b978-0-12-185287-0.50023-5 ◽

1994 ◽

pp. 320-339

Author(s):

Barry W. Hicks ◽

Kimon J. Angelides

Keyword(s):

Fluorescence Microscopy ◽

Ion Channel ◽

Channel Dynamics ◽

Living Neurons

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Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

Infection and Immunity ◽

10.1128/iai.00552-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5617-5624 ◽

Cited By ~ 7

Author(s):

Fang Cai ◽

Carrie B. Adrion ◽

James E. Keller

Keyword(s):

Culture Medium ◽

Reaction Model ◽

Cultured Neurons ◽

Total Inhibition ◽

Botulinum Neurotoxins ◽

A Cell ◽

Proteolytic Activities ◽

Living Neurons ◽

Subsequent Addition

ABSTRACT Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

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