Abstract
The localization of ion channels on myelinated axon is closely related with the saltatory conduction of action potential (AP). Abnormal changes in these channels contribute to multiple mental diseases. The development of cryo-Electron Tomography (cryo-ET) has provided a promising prospect for peering into ion channels in their native environment at high resolution. Previous achievements are reviewed here on cryo-ET. Accordingly, a cryo-ET workflow is designed for understanding ion channels localization in myelinated axon, especially nodes of Ranvier, which are significant for the saltatory conduction involved in the propagation of high-speed AP. The workflow is divided into six parts: the preparation of neural cultures with myelin, antibodies and immunofluorescence staining, frozen-hydrated sample preparation, cryo-ET imaging, cryo-correlative light and electron microscopy (cryo-CLEM) imaging, three-dimensional (3D) reconstruction and refinement. The purpose is to conceive a possible solution for the problems related to ion channel compounds including localization, conformation dynamics, accessory structures of ion channel and transient regulatory factors, and thus provide insights into treating neurological diseases caused by abnormal ion channels activity.
Highlights
The normal function of the brain relies on accurate and rapid conduction of electrical signal, which is called action potential (AP)
This paper aims to review antecedent achievements, design a workflow for mapping ion channels at the nodes of Ranvier to resolve the events underlying the saltatory conduction, and determine the still missing parts of ion channel complex as the possible topics of future exploration, for reference in treating neurologic diseases either by means of drugs or neural stem cell transplantation
Nodes of Ranvier are bordered by the paranodal axoglial junctions (PNJ), a specialized axon-glial contact formed between the axolemma and the paranodal loops which are formed by myelin lamellae and closely opposed to the axon with a gap of only 2.5 - 3 nm [3]
Summary
The normal function of the brain relies on accurate and rapid conduction of electrical signal, which is called action potential (AP). Abnormality in the permeability, expression level or electrical excitability of sodium voltage-gated channels assembled with the first or sixth type of α-subunit, namely Nav1.1 or Nav1.6 channels, at nodes of Ranvier lead to autism, epilepsy syndromes, fibromyalgia and periodic paralysis [5]; and the pathogenesis of myokymia and benign familial neonatal convulsions is closely related to mutant potassium voltage-gated channel subfamily Q member 2 (KCNQ2) and member 3 (KCNQ3) channels [6]. Researches on the synaptic activities or localization and regulation process of ion channels on myelinated axons have provided valuable insights into the mechanism of mental disorders and potential therapeutic targets, especially with recent optimizations of data acquisition and processing in cryo-electron tomography to obtain the three-dimensional information at subnanometer resolution [8] [9] from exquisitely preserved frozen-hydrated specimens in a close-to-life state. This paper aims to review antecedent achievements, design a workflow for mapping ion channels at the nodes of Ranvier to resolve the events underlying the saltatory conduction, and determine the still missing parts of ion channel complex as the possible topics of future exploration, for reference in treating neurologic diseases either by means of drugs or neural stem cell transplantation
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