Voltage-gated sodium channels (VGSCs), also known as voltage-dependent sodium channels (VDSCs), are transmembrane proteins that play important roles in the electrical signaling of cells ^[1]. Mammalian Nav channels are formed by a large pseudotetrameric pore-forming α subunit (260 kDa) that associates with one or two β subunits (30–40 kDa). In contrast, prokaryotic Nav channels that have been used to explore the structure of eukaryotic Navs are formed by homotetramers. Nav channel α subunits show tissue specific expression profiles. Nav1.1, Nav1.2, and Nav1.3 subtypes are expressed in the central nervous system (CNS). Nav1.6 is expressed in both the peripheral and central nervous system, whereas Nav1.7, Nav1.8, and Nav1.9 are mostly restricted to the peripheral nervous system (PNS) ^{[2, 3]}. Nav1.4 and Nav1.5 channels are abundant in skeletal and cardiac muscles, respectively ^[4].

VGSCs consist of an α subunit, which can be coupled to one or two β subunits. In humans there are nine different α subunits, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, and Nax. The different α subunits define the distinct Nav channel subtypes and contain the receptor sites for drugs and toxins that act on Nav channels. The α subunits are large, single-chain polypeptides composed of approximately 2000 amino acid residues organized in four homologous domains, designated DI to DIV, that form a pseudotetrameric structure. The sequence homology of mammalian Nav subtypes is very high, being greater than 50% in the transmembrane and extracellular domains ^[2]. Each domain is composed of six transmembrane helical segments named S1 to S6. In contrast, prokaryotic Nav channels are far simpler, consisting of homotetramers of four identical polypeptide chains. Each polypeptide chain contains six transmembrane segments (S1–S6) and exhibits high sequence homology when compared to eukaryotic domains ^[4]. Segments S1–4 of Nav channel α subunits from each domain form the VSD, an important structural module which function is the regulation of channel opening upon depolarization of the membrane. The flexibility of the VSD is primarily mediated by movement of positively charged arginine and lysine residues positioned at every third residue within each S4 helix. The four voltage-sensing domains are arranged around a central aqueous channel formed by the pore domain (PD). The VSD is connected to the PD by an intracellular linker between transmembrane segments S4 and S5. Upon depolarization, the positively charged S4 transmembrane segments are believed to move toward the extracellular surface. This motion is transferred to the pore domain via intracellular linkers, causing a conformational change that results in the opening of the channel pore. During depolarization, the channel inactivates as the inactivation gate (vide infra) folds into the channel pore. Upon membrane repolarization, Nav channels recover from inactivation and the S4 segments return to their resting positions, becoming available for the next depolarization. From a study of the functional contributions of the amino acid residues at the VSD of skeletal muscle channel subtype Nav1.4, Ahern and co-workers found that the highly conserved aromatic side chain at the S2 hydrophobic core makes distinct functional contributions in each of the four Nav domains. This study showed that, surprisingly, not all of the four S1–S4 structures contributing to the VSD adopt the same structural conformation at a specific Nav channel state ^[5]. Segments S5, S6, and the extracellular connecting pore-loops (P-loops) form the channel pore and the selectivity filter (SF). The SF is invariantly composed of aspartate (D) in DI, glutamate (E) in DII, lysine (K) in DIII, and alanine (A) in DIV (or DEKA), forming the geometrically narrowest region of the ion pore, which is called the constriction site or inner ring and selectively allows the flux of hydrated Na⁺ through the ion pore ^[4]. There are four β subunits, Navβ1, Navβ2, Navβ3, Navβ4 ^[6].

Neurotoxin receptor site 5 binds the complex polyether toxins brevetoxin and ciguatoxin, which are made by dinoflagellates and cause toxic red tides in warm ocean waters. Transmembrane segments IS6 and IVS5 are implicated in brevetoxin binding from photoaffinity labeling studies. Neurotoxin receptor site 6 binds δ-conotoxins, which slow the rate of inactivation like the α-scorpion toxins ^[7]. The location of neurotoxin receptor site 6 is unknown. Finally, the local anesthetics and related antiepileptic and antiarrhythmic drugs bind to overlapping receptor sites located in the inner cavity of the pore of the sodium channel ^[7].

The AK-42 inhibitor is located above the Sext site and blocks the Cl⁻ channel. The hydrophobic pocket was formed by M460, F252, F459, F463, F306, and L397. Moreover, the AK-42 heterocycle also had hydrophobic interactions with I112 and L116 ^[8].

Molecular dynamics simulations was uesd to show that PI(4,5)P2 binds stably to an inactivated structure of Nav1.4 at a conserved site within the DIV S4-S5 linker, which couples the voltage sensing domain (VSD) to the channel pore ^[9].

Nav1.7 The transition of S6IV and rearrangement of the gating residues (Black Sphere in the 3D structure viewer), such as Leu398, Leu964, Ile1457 and Ile1756 was binging site of bupivacaine (BPV), lacosamide (LCM-1), and carbamazepine (CBZ). This site is constituted by the cytosolic residues on the S6 tetrahelical bundle that are adjacent to the gating residues. Although the three drugs completely overlap in site BIG, the coordinating details vary BPV is primarily surrounded by hydrophobic residues, including Leu964, Leu967, Leu968, and Phe971 from S6II, Ile1457 from S6III, and Ile1756 and Leu1760 from S6IV ^[7]. Kidocaine binding to the channel enhances PF-05089771 inhibition by altering the equilibrium between resting states (with D4S4 in the inner position) and inactivated states (with D4S4 in the outer position) ^[10].

polypeptide toxins alter channel gating by voltage-sensor trapping through binding to extracellular receptor sites, and this toxin interaction has now been modeled at the atomic level for a β-scorpion toxin ^[11].

Neurotoxin receptor site 1 binds the nonpeptide pore blockers tetrodotoxin (TTX) and saxitoxin and the peptide pore blocker μ-conotoxin ^[15]. The receptor sites for these toxins are formed by amino acid residues in the pore loops and immediately on the extracellular side of the pore loops at the outer end of the pore.

Site-directed mutagenesis to determine the contribution to activity of Leu-18 is the most significant single contributor to the high affinity of Anthopleurin B identified to date ^[16]. Bulleyaconitine A is located around domain I-II fenestration (site-2 neurotoxin binding site), it partially blocks Nav1.3 path and expands the pore-lining helices. ICA121431 preferentially binds to activated domain IV voltage-sensor, consequently strengthens the Ile-Phe-Met motif binding to its receptor site ^[17]. Both hydrophilic low molecular weight toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance ^[18]. Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6, and Nav1.7 are blocked by low nanomolar concentrations of TTX; therefore, these subtypes are classified as TTX-sensitive, whereas, Nav1.5, Nav1.8, and Nav1.9 are inhibited by only high micromolar TTX concentrations.

Neurotoxin receptor site 2 binds a family of lipid-soluble toxins, including batrachotoxin, veratridine, aconitine, and grayanotoxin, which enhance activation of sodium channels. Photoaffinity labeling and mutagenesis studies implicate transmembrane segments IS6 and IVS6 in the receptor site for batrachotoxin. Neurotoxin receptor site 3 binds the α-scorpion toxins and sea anemone toxins, which slow the coupling of sodium channel activation to inactivation. These peptide toxins bind to a complex receptor site that includes the S3-S4 loop at the outer end of the S4 segment in domain IV. Neurotoxin receptor site 4 binds the β-scorpion toxins, which enhance activation of the channels. The receptor site for the β-scorpion toxins includes the S3-S4 loop at the extracellular end of the voltage-sensing S4 segments in domain II ^[7].

Pure hoiamide A potently inhibited [3H]batrachotoxin binding to voltage-gated sodium channels (IC50 = 92.8 nM) and activated sodium influx (EC₅₀ = 1.73 μM) in mouse neocortical neurons, as well as exhibited modest cytotoxicity to cancer cells. Further investigation revealed that hoiamide A is a partial agonist of site 2 on the voltage gated sodium channel ^[12]. Four hydrophobic amino acid residues, one from each intracellular end of the S6 segments, form a small intracellular cavity named the activation gate. The putative activation gate in wild-type Nav1.7 channels consists of four aromatic residues, one from each S6 helix (Y405 in DI, F960 in DII, F1449 in DIII, and F1752 in DIV). Using structural modeling-guided mutagenesis, Waxman and colleagues have demonstrated that when the L955 residue in DIVS6 is deleted, the orientation of F960 shifts radially toward the S6 of DIII, disrupting the activation gate ^[13]. Deletion of L955 of human Nav1.7 channels from a family with inherited erythromelalgia causes a robust hyperpolarizing shift (25 mV) in the voltage dependence of activation ^[14].

Positive allosteric interaction between the Antillatoxin (ATX) binding site and neurotoxin site 5 of Nav1.4 α-subunit ^[19]. Hydrophobic alkaloid toxins and related lipid-soluble toxins act at intramembrane sites and alter voltage-dependent gating of sodium channels via an allosteric mechanism ^[20].

It has been reported that the sensitivity of the receptor site 3 can be enhanced by specific ligands of another receptor site. For instance, brevetoxin (Pbtx-1) bound to receptor site-5 has positive allosteric modulation on LqhαITa site-3 toxin binding to insect sodium channels^{[21, 22]}.