Binding to voltage-gated sodium channel

Due to their critical role in neuronal function, sodium channels are known molecular targets of neurotoxins and neurotoxicants (Caterall et al., 2012; Wakeling et al., 2012). The essentiality of sodium channels in nerve conduction comes from classic literature on tetrodotoxin (TTX). TTX is a sodium channel blocker that inhibits the firing of action potentials in neurons by binding to the voltage-gated sodium channels (VGSC/NaV) in nerve cell membranes. This action blocks the passage of sodium ions into the neuron, ions responsible for the rising phase of an action potential (AP). There is strong evidence implicating a similar TTX-like of pyrethroid insecticides on VGSC. This block of VGSC is supported by an extensive body of literature on the action of pyrethroid insecticides on mammalian sodium channels. Binding studies using radioactive pyrethroid demonstrated specific binding of the pyrethroid to rat brain VGSC α subunits (Trainer et al., 1997).

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 across the membrane along their electrochemical gradient. Ion channels 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 of both types are essential for chemical communication between cells, i.e., synaptic transmission. Ion channels also function to maintain membrane potential and initiate AP to propagate electrical impulses. VGSC are therefore responsible for AP initiation and propagation in most excitable cells, including nerve, muscle and neuroendocrine cell types. It is important to note that functional VGSC are present in both grey and white matter in the brain. Approximately 50% of white matter oligodendrocyte precursor cells receive synaptic inputs and can produce trains of VGSC-dependent APs (Fields, 2008). VGSC are also present on microglia where they contribute to the 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 tissue-, region- and time- specific manners. 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, their sensitivity and their functional role, all contribute to the manifestation of toxicity and age-dependent sensitivity, of chemicals acting at this site.

Interaction of compounds with VGSC can be measured directly with radioligand binding (Trainer et al 1997), while the expression and localization of VGSC on different cell types can be assessed using immunohistochemical methods. The following discussion focuses on interactions between VGSC and pyrethroids, but similar data exist for other compounds that bind to VGSC. Several other approaches provide indirect evidence of interactions of chemicals with VGSC. The published literature contains hundreds of reports identifying point mutations in VGSC that alter both the effects on the channel as well as the sensitivity to pyrethroid toxicity. Both increased and decreased modification of the insect and mammalian VGSC by pyrethroids have been demonstrated, specific action dependent on the location and type of point mutations (e.g. Vais et al., 2000; 2001). Finally, the demonstration of stereo-specific effects of the pyrethroids on binding (Soderlund 1985; Brown et al., 1988) as well as electrophysiological responses (Narahashi 1982; Narahashi1996; Narahashi., 2000; Narahashi., 2002) also supports interaction of VGSC and pyrethroids. A model for binding of pyrethroids in insect VGSC has been developed (O’Reilly et al., 2006). Together, these observations provide strong evidence of pyrethroid binding to VGSC (for additional review, see Field et al 2017).

VGSCs are present in many different cell types of the nervous system (NS), including neurons, oligodendrocytes, Schwann cells (Baker, 2002; Jessen and Mirsky, 2005; Ritche, 1992; Chiu, 1991) and microglia (Jung et al., 2013; Black and Waxman reviewed in Hossain et al., 2017; Paez et al., 2009; Berret et al., 2017).

Moreover, every cell within living organisms actively maintains a low intracellular sodium concentration that is 10–12 times lower than the extracellular concentration. The cells then utilize this transmembrane sodium concentration gradient as a driving force to produce electrical signals, and if the driving force is sufficiently strong, an AP is produced. The protein family comprising VGSC (Navs) is essential for such signaling and enables cells to change their electrical status in a regenerative manner and to rapidly communicate with one another. The existence of VGSC was first predicted from studies of electrical activity in squid giant axon and later identified through molecular studies in the electric eel. Since then, these proteins have been observed in organisms ranging from bacteria to humans (Chaihne, 2018).

Sodium channels consist of highly processed α subunit, which is approximately 260 kDa, associated with auxiliary β subunits of 33–39 kDa. Sodium channels in the adult CNS and heart contain a mixture of β1–β4 subunits, while sodium channels in adult skeletal muscle have only the β1 subunit. Nine different VGSC have been identified using electrophysiological recording, biochemical purification, and cloning (Catterall, 2007; Catterall, 2012).

Nomenclature of the different sodium channel alpha (pore-forming) subunits is based on 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).  (Catterall, 2012).

In mammals, numerous 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 impairs nervous system structure and function, disrupts muscle function, pain reception, and cardiac rhythm (Chahine, 2018). VGSCs show complex regional and temporal ontogeny in mammals. Table 1, from Shafer et al., 2005 provides an overview about the alpha subunits and their developmental and tissue expression pattern. Pyrethroid interactions with Nav1.1 (James et al., 2017), Nav1.3 (Meacham et al., 2008; Tan and Soderlund 2009), Nav1.6 (Tan and Soderlund, 2010), Nav1.7 (Tan and Soderlund, 2011) and Nav1.9 (Nutter and Cooper, 2014; Bothe et al., 2021) channels.

ß1b and ß3 expression is high during prenatal and early postnatal period in nervous system mammals, followed by increased expression of ß1, ß2 and ß4 in the first postnatal week which then persists through adulthood. While different cell types in the brain express different ß subunits, the ß1 subunit is ubiquitously expressed with moderate heterogeneity. Its subcellular localization provides specific functionalities, e.g. high density of ß1 at the nodes of Ranvier modulates surface expression and gating of the VGSCα subunit  while in the paranodal region ß1 mediates axonal-glial cell adhesion. The ß2 protein shares some similar expression pattern with ß1 and appears to provide responsiveness to inflammatory and neuropathic pain in the peripheral nervous system (PNS). In contrast ß3 mRNA and protein are expressed ubiquitously thought the developing CNS and in adult mice it is greatly reduced except for some structures like the hippocampus. This differs in human brain, where ß3 remains highly expressed throughout adulthood. The expression profile of ß4 is mostly restricted among the ß subunits, and often related to neurons with spontaneous or burst firing APs.  Finally, β subunits are also expressed in various glia where they may function as cell adhesion guides and cues for neurodevelopment, including coordinating neurite outgrowth, axonal fasciculation, and neuronal migration (Hull et Isom 2018). Importantly, co-expression of β subunits with the α subunit modulates the function of the α subunit and can influence the binding of various ligands to the α subunit (Tan et al., 2011). In general, embryonically expressed forms of VGSCs are replaced by expression of adult forms as neurodevelopment proceeds.

Due to this complex ontogeny of VGSCs it is currently not possible to specify which VGSCs subtypes and which developmental stages are particularly essential and thus important for this AOP.

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