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Key Event Title
Inhibit, voltage-gated sodium channel
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Molecular events lead to epilepsy||KeyEvent||Allie Always (send email)||Open for adoption|
|presynaptic neuron 1 activation to epilepsy||KeyEvent||Brendan Ferreri-Hanberry (send email)||Open for adoption|
|Inhibition of voltage gate during development is leading to cognitive disorders||MolecularInitiatingEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite|
|All life stages|
Key Event Description
Ion channels are integral membrane proteins that are critical for neuronal function. They form pores in the plasma membrane that allow certain ions to travel with their concentration gradient across the membrane. Those that open in response to a change in membrane voltage potential are called voltage-gated ion channels. Channels that open in response to binding using a chemical signal or molecule are ligand-gated ion channels. In neurons, ion channels are essential for chemical communication between cells, or synaptic transmission. Ion channels also function to maintain membrane potential and initiate and propagate electrical impulses. Voltage-gated sodium channels are therefore responsible for action potential initiation and propagation in excitable cells, including nerve, muscle and neuroendocrine cell types. They are also expressed at low levels in non-excitable cells. It is important to note is that functional VGSC are present in both grey and white matter in the brain and approximately 50% of white matter oligodendrocyte precursor cells producing trains of action potentials and receiving synaptic input (Fields, 2008). VGSC are also present on microglia cells and contribute to release of major pro-inflammatory cytokines (Hossain et al., 2017).
Mammalian VGSC are composed of one α and two β subunits. Ten separate α subunits (Ogata and Ohishi, 2002) and four different β subunits (Isom, 2002) have been identified and are expressed in a tissue, region and time specific manner. The diverse functional roles of VGSCs depend on the numerous potential combinations of α and β subunits (Ogata and Ohishi, 2002). The type of VGSCs expressed in different cell types and regions, and their sensitivity and their functional role, may all contribute to the manifestation of toxicity and age dependent sensitivity, including the effects caused by pyrethroids.
At resting membrane potentials the channel is closed. During the rising phase of an action potential the channel activates or opens. Channel inactivation contributes to the falling phase. During the undershoot phase the channel deactivates before returning to the closed phase once resting membrane resting potential has been restored. Source: adapted from Motifolio Biomedical PowerPoint Toolkit Suite.
How It Is Measured or Detected
The sodium channel protein has been discovered and characterised in biochemical and molecular detail, even to atomic resolution. The initial works performed to measure and detect the electrical signals in nerves were initiated by Hodgkin and Huxley in 1952, showing a voltage‐dependent activation of sodium current that carries Na+ inward. The structure of VGSCs is nowadays known in detail and some seminal papers are available (Catterall, 2012).
Intracellular microelectrode recording using voltage or patch clamp are the common methods used for electrophysiological studies of VGSC. Channels and locations can also be measured using immunohistochemical methods, transcriptomics and at protein levels.
Expression of different sodium channel isoforms can be measured using a panel of sodium channel subunit-specific antibodies. Quantification of immunocytochemical staining is difficult due to differences in equipment, tissue preparation, inter-assay variability and analysis methods. However, using a quantitative approach, it is possible to determine the localisation and relative levels of sodium channel subunit protein expression (Westenbroek et al., 2013). PCR amplification and competitive PCR approach, real-time PCR, are used to isolate the mRNA levels of VGSC isoforms (Haufe et al., 2005).
Domain of Applicability
Every cell within living organisms actively maintains an intracellular Na+ concentration that is 10–12 times lower than the extracellular concentration. The cells then utilise this transmembrane Na+ concentration gradient as a driving force to produce electrical signals, sometimes in the form of action potentials. The protein family comprising VGSC (Navs) is essential for such signalling and enables cells to change their status in a regenerative manner and to rapidly communicate with one another. VGSC were first predicted in squid and were later identified through molecular biology in the electric eel. Since then, these proteins have been discovered in organisms ranging from bacteria to humans (Chaihne, 2018).
Sodium channels consist of a highly processed α subunit, which is approximately 260 kDa, associated with auxiliary β subunits of 33–39 kDa. Sodium channels in the adult central nervous system (CNS) and heart contain a mixture of β1–β4 subunits, while sodium channels in adult skeletal muscle have only the β1 subunit. Nine different sodium channels have been identified using electrophysiological recording, biochemical purification, and cloning (Catterall, 2012).
Nomenclature of the different sodium channels utilises a numerical system to define subfamilies and subtypes based on similarities between the amino acid sequences of the channels. In this nomenclature system, the name of an individual channel consists of the chemical symbol of the principal permeating ion (Na) with the principal physiological regulator (voltage) indicated as a subscript (Nav). The number following the subscript indicates the gene subfamily (currently only Nav 1), and the number following the full point identifies the specific channel isoform (e.g. Nav 1.1). This last number has been assigned according to the approximate order in which each gene was identified. Splice variants of each family member are identified by lower-case letters following the numbers (e.g. Nav 1.1a). Nine mammalian sodium channel isoforms have been identified and functionally expressed with all greater than 50% identical in amino acid sequence in the transmembrane and extracellular domains, where the amino acid sequence is similar enough for clear alignment (Catterall, 2012). In addition to these nine sodium channels that have been functionally expressed, closely related sodium channel-like proteins (Nax) have been cloned from mouse, rat and human. They are approximately 50% identical to the Nav 1 subfamily of channels but more than 80% identical to each other (Catterall, 2012).
In mammals, neuronal VGSC are expressed in the adult and developing brain. Evidence from mutation and knockout animal models demonstrates that perturbation of VGSC function during development impair nervous system structure and function, including muscle function, pain reception and cardia arrythmias (Chahine, 2018). VGSC show complex regional and temporal ontogeny in mammals (see Table 1, from Shafer et al., 2005). In general, embryonically expressed forms of VGSCs are replaced by expression of adult forms as neurodevelopment proceeds.
This complex ontogeny of VGSCs confounds any simple linkage of VGSCs to adverse outcomes and is an uncertainty in the development of this AOP. Since brain development in both humans and rodents extends from early gestation through lactation, it is not currently possible to state which VGSC subtype, or subtypes, may be responsible for the AOs.
Ion channels, including VGSCs, are also expressed in oligodendrocytes, Schwann cells (Baker, 2002) and microglia (Hossain et al., 2017). The expression and function of VGSS in cells of the oligodendrocyte lineage follow a time and regional ontogeny. While present and active in the early stages of oligodendrocyte maturation, VGSC function decreases over developmental time and is absent in mature oligodendrocytes (Paez et al., 2009; Berret et al., 2017). Knockdown of VGSC in rat oligodendrocyte precursor cells (OPCs) leads to reduced myelination suggesting a function of VGSC for axon myelination (Berret et al., 2017).
The physiological and anatomical ontogeny of Schwann cells is well known (Jessen and Mirsky, 2005). VGSCs are present in Schwann cells including the tetrodotoxin sensitive and Nav 1.7 types (Ritche, 1992; Chiu, 1991; Baker, 2002) less is known about their developmental profile.
Microglia cells express several ion channels, including Cl-, K+, H+ and Ca2+ and VGSC that are involved in several cellular functions such as maintaining the membrane potential, cellular volume and intracellular ion concentrations. VGSCs are demonstrated, to be present both in rodents and human microglia. Different isoforms are present in primary microglia (Nav 1.1, 1.2, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.1 isoforms) compared to immortalised BV2 cells (Nav 1.2, 1.3, 1.4, 1.6, 1.8, 1.9, and 2.1 isoforms) (Jung et al., 2013; Black and Waxman, 2012; reviewed by Hossain et al., 2017). Presence of sodium channel isoforms in immortalised BV2 cells and primary microglia were detected by mRNA expression with standard PCR. BV2 cells express some sodium channel isoforms including Nav 1.2, 1.3, 1.4, 1.6, 1.8, 1.9, and 2.1 whereas primary microglia from 1–2-day-old mice express channel isoforms Nav 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 1.8, 1.9, and 2.1. Primary microglia expressed higher levels of Nav 1.1. 1.2, 1.3, 1.6, 1.9, and 2.1 compared with BV2 cells.
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Berret E, Barron T, Xu J, Debner E, Kim EJ and Kim JH, 2017. Oligodendroglial excitability mediated by glutamatergic inputs and Nav1.2 activation. Nature Communications, 8(1), 1–15. https://doi.org/10.1038/s41467–017–00688–0
Black JA and Waxman SG, 2012. Sodium channels and microglial function. Experimental Neurology, 234(2), 302–315. https://doi.org/10.1016/j.expneurol.2011.09.030
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