Sodium channels in non-excitable cells: Powerful actions and therapeutic targets beyond Hodgkin and Huxley

Abstract

Voltage-gated sodium channels (VGSCs) are best known for their role in the generation and propagation of action potentials in neurons, muscle cells, and cardiac myocytes which have traditionally been labeled as “excitable”. However, emerging evidence challenges this traditional perspective. It is now clear that VGSCs are also expressed in a broad spectrum of cells outside the neuromuscular realm, where they regulate diverse cellular functions. This article summarizes current knowledge on the expression, regulation, and function of VGSCs in non-neuromuscular cells, highlighting their contributions to physiological processes and pathological conditions. Dynamic expression patterns of VGSCs in different cell types, involvement of VGSCs in cellular functions such as phagocytosis, motility, and cytokine release, and their potential as therapeutic targets for diseases that include inflammatory disorders, osteoarthritis, and cancer, are discussed. This new understanding of VGSCs and their effects on cells outside the neuromuscular realm opens new avenues for research and therapeutic interventions.

Voltage-gated sodium channels in non-excitable cells

The groundbreaking work of Hodgkin and Huxley [1] unveiled the fundamental role of voltage-gated sodium channels (VGSCs) in generating action potentials in excitable cells such as neurons, muscle cells, and cardiomyocytes [2-5]. VGSCs are transmembrane protein complexes composed of a central pore-forming α-subunit and smaller β-subunits. Nine α-subunit isoforms (Nav1.1-Nav1.9, sometimes characterized as Tetrodotoxin-sensitive (TTX-S): Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6, Nav1.7; or Tetrodotoxin-resistant (TTX-R): Nav1.5, Nav1.8, Nav1.9) have been identified in mammals, each with unique expression pattern and voltage-dependent gating and kinetics [6]. Despite these differences, all VGSCs share a conserved architectural motif, featuring four homologous domains, each containing six transmembrane segments. The selectivity filter within the VGSC pore discriminates between sodium and other ions, facilitating rapid sodium influx upon membrane depolarization. In cells containing a sufficient number of VGSCs this influx triggers a regenerative feedback loop underlying the rapid upstroke of the action potential. Subsequently, VGSCs transition to an inactivated state that terminates the action potential. This intricate process of voltage-dependent activation, selectivity, and inactivation is crucial for the precise timing and fidelity of electrical signaling in excitable cells.

The canonical role of VGSCs is firmly established for cells such as neurons, myocytes, and cardiomyocytes, whose major function involves action potentials produced by high densities of channels [2-5]. However, it is becoming increasingly clear that VGSCs are present and functional in multiple types of cells that are traditionally not considered to be excitable, where they are expressed at low densities [7]. As an extreme example, a chondrocyte contains 0.1 channels/μm² [8], while in mammalian neurons, the Nav channel density at impulse trigger zones is 1,000-5,000-fold higher [9]. Remarkably, despite their low density, these channels have strong effects on the biology of chondrocytes [8]. Accumulating evidence indicates that VGSCs play important roles in regulating multiple effector functions including motility, phagocytosis, and cytokine release in an increasing array of cells that have traditionally been considered as non-excitable (here and thereafter the term “non-excitable” is used for cell-types other than those classically labeled as excitable - neurons, muscle cells and cardiac myocytes - where action potential electrogenesis has been most intensively studied). Emerging evidence suggests that the dysregulation of VGSCs in non-excitable cells can contribute to the pathogenesis of multiple disorders, including inflammatory disorders, osteoarthritis and cancer (Table 1).

Table 1.

VGSCs in non-excitable cells: expression, impact on cell function and suggested therapeutic implications.

Cell type	VGSC mRNA and/or protein	Functional expression	Impact on cell function	Possible therapeutic implications: potentially efficacious drugs suggested in literature	References
Astrocytes	Nav1.2, Nav1.3, 1Nav1.5, Nav1.6	2TTX-S, 3TTX-R	Na+/K+-ATPase regulation; ionic homeostasis, regulation of glial scarring	Multiple sclerosis (MS), epilepsy	[13-20, 25, 29, 30, 142]
Oligodendrocytes	Nav1.1, Nav1.2, Nav1.3, Nav1.8	TTX-S	Cell process extension, glia-axon interactions, proliferation, migration, differentiation	Promote remyelination, e.g. in MS	[33, 35, 36, 143, 144]
Schwann cells	Nav1.2, Nav1.3, Nav1.6	TTX-S	Unknown	Unknown	[20, 37-39]
Muller glial cells	Nav1.6, Nav1.9	TTX-S, TTX-R	Regulation of cell volume, gliotransmitter release, proliferation	Retinal diseases	[108, 145-148]
Dendritic cells and macrophages	Nav1.5, Nav1.6, Nav1.7	TTX-S (DC)	Phagocytosis, migration, cytokine production	Inflammatory disorders, Phenytoin	[40-43, 149]
Microglia	Nav1.1, Nav1.5, Nav1.6	TTX-S, TTX-R	Phagocytosis, migration, release of pro-inflammatory cytokines	Neuroinflammation, MS Phenytoin, Carbamazepine, Flecainide and Safinamide	[43-49, 150]
Lymphocytes	Nav1.2, 1.3, 1.4, 1.6, 1.7 and 1.9 in hB-cells; Nav1.5 and Nav1.9 in hT “Jurkat” cell line	TTX-S, TTX-R	Maintenance of resting membrane potential, regulation of intracellular Ca2+, T-cell differentiation	T-cell leukemia	[117-119, 151-153]
Neutrophils	Nav1.1, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7	4N.A.	Adhesion, transmigration, chemotaxis	Ischemia-reperfusion injury. Lidocaine	[120-124]
Erythrocytes	Nav1.4, Nav1.7	N.A.	Unknown	Unknown	[125]
Endothelial cells	Nav1.2, Nav1.5, Nav1.6, Nav1.7, Nav1.9	TTX-S, TTX-R	Angiogenesis, proliferation, chemotaxis, vasomotor tone, response to shear stress	Cardiovascular and pulmonary diseases, hypertension	[50, 51, 53, 55]
Retinal Epithelium Cells	Nav1.1, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7 Nav1.8; Nav1.9	TTX-S, TTX-R	Phagocytosis of photoreceptor outer segments, cell-cell communication	Age-related macular degeneration	[126-128, 154, 155]
Enterochromaffin cells	Nav1.3	TTX-S	5-HT release, AP firing	Visceral pain, irritable bowel syndrome	[56-60]
Adrenal chromaffin cells	Nav1.3	TTX-S	Catecholamine release, AP firing	Unknown	[110-112]
Fibroblasts	Nav1.2, Nav1.3, Nav1.5, Nav1.6, Nav1.7	TTX-S, TTX-R	Cell motility, volume regulation	Atrial fibrillation, fibrotic lung disease	[52, 62-64]
Keratinocytes	Nav1.1, Nav1.2, Nav1.6, Nav1.7, Nav1.8, Nav1.9	N.A.	ATP release, nociceptor sensitization	CRPS, post-herpetic neuralgia, psoriasis	[129-132, 156]
Pancreatic β-cells	Nav1.3, Nav1.6, Nav1.7; β1	TTX-S	Generation of APs, insulin secretion	Diabetes. Carbamazepine	[66, 67, 69, 71, 73-75]
Mesenchymal Stem Cells	Nav1.2 in rat; Nav1.7 in human	TTX-S	Cell proliferation and migration, transition to cancer	N.A.	[133-136, 157]
Osteoblasts	Nav1.2, Nav1.3, Nav1.6, Nav1.7	TTX-S	Cellular signaling and communication (APs), osteogenic differentiation	Osteoporosis, bone health (fractures). Carbamazepine and Phenytoin (negative impact)	[76-80, 157]
Odontoblasts	Nav1.1-Nav1.9	TTX-S, TTX-R	Sensory transduction, dentinogenesis	Dental pain	[137-139, 158]
Chondrocytes	Nav1.2, Nav1.3, Nav1.4, Nav1.6, Nav1.7, Nav1.9	TTX-S, 5ProTx-S	Regulate chondrocyte secretome and intracellular ion homeostasis, differentiation, proliferation, cartilage development	Osteoarthritis. Carbamazepine and PF-04856264	[8, 81-84, 86]
Cancer cells	Nav1.4, Nav1.5, Nav1.6, Nav1.7	TTX-S, TTX-R	Cell migration and invasion, metastasis, cell proliferation	Breast, colon, lung, prostate, pancreatic, ovarian, endometrial cancer; glioblastoma. Naringenin, ranolazine, lidocaine	[89, 90, 92-97, 99-105, 141, 159-162]

¹Nav channels specifically implicated in pathological condition are highlighted in Bold

²TTX-S currents: Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6, Nav1.7

³TTX-R currents: Nav1.5, Nav1.8, Nav1.9

⁴N.A. data not available

⁵ProTx-S, current sensitive to ProTx-II (20 nM), Nav1.7-selective blocker

This review highlights the expanding understanding of VGSCs in non-excitable cells. The article first describes recent studies on non-excitable cell-types where evidence for VGSCs is strongest, exploring their functional significance, molecular mechanisms, contributions to disease pathobiology, and potential as therapeutic targets. Then common themes in expression of VGSCs in these cells are discussed, and questions for the future are posed.

Dynamic expression of VGSCs in non-excitable cells

The expression of VGSCs within cells that have not been traditionally considered to be electrically excitable was initially demonstrated via in situ hybridization or RT-PCR (mRNA), immunohistochemistry (protein) or patch-clamp (ionic currents). Data summarized in ARCHS4 (Figure 1), an online RNA-seq data resource [10], underscore the presence of VGSCs in multiple cell-types, in a variety of physiological functions and in multiple disease phenotypes. Different expression levels and different mRNA splicing patterns of VGSC genes during development and with changes in experimental conditions or disease state, imply that the effects of VGSC on cell function can change over time [11, 12]. This article focuses first on cell-types where VGSC expression has been especially well-studied and where functional aspects of VGSC expression is especially well-understood.

Figure 1. Tissue expression pattern of VGSCs.

Montage image of data for Nav1.1 - Nav1.9 expression within different tissues, adapted from the ARCHS4 web resource (https://amp.pharm.mssm.edu/archs4/gene/SCN1a) showing SCN1a, SCN2a, etc. expression in various tissues. All RNA-seq and ChIP-seq sample and signature search (ARCHS4) (https://maayanlab.cloud/archs4/) is a resource that provides access to gene and transcript counts uniformly processed from all human and mouse RNA-seq experiments from the Gene Expression Omnibus (GEO) and the Sequence Read Archive (SRA). The ARCHS4 website provides the uniformly processed data for download and programmatic access.

Astrocytes

VGSC expression in astrocytes has been especially well-studied. Astrocytes play a multifaceted role in the CNS, where they regulate the extracellular environment surrounding neurons and synapses and contribute to brain development and repair. Astrocytes express Nav1.2, Nav1.3, Nav1.5 and Nav1.6 sodium channels [13-20]. Early patch-clamp studies in vitro [13] revealed the presence of multiple types of functional VGSCs in these cells. (Figure 2B) [14]. Notably, despite a lower VGSC density than in neurons, action potential-like responses can be evoked in some astrocytes if their membrane potential is hyperpolarized to remove resting inactivation [14]. Patch-clamp studies on hippocampal slices demonstrated that VGSCs are present not just in culture but, on the contrary, are present in astrocytes within their native CNS environment [14, 21].

Figure 2. Sodium channels in astrocytes.

(A) Nav1.5 expression in astrocytes within multiple sclerosis tissue. An astrocyte within a normal white matter (WM) exhibits minimal Nav1.5 labelling (top panel). In contrast, an astrocyte in the WM between two areas of macrophage infiltration is hypertrophic and shows upregulation of Nav1.5 (panel 2). Astrocytes at the border of (panel 3) and within (panel 4) the active lesion display intense Nav1.5 immunolabelling. Modified from [19]. (B) In recordings from spinal cord astrocytes, voltage steps more positive than −50 mV activate sodium currents. Both TTX-S and TTX-R sodium currents were present (right panel). Modified from [15]. (C) In an in vitro model of the glial response to injury, astrocytes grow together following scratch injury, resulting in closure of the wound compared to the average original wound size (black arrows) after a 24 h time period. Block of VGSCs with TTX or block of reverse sodium-calcium exchange with KB-R7943 attenuate closure of the scratch wound. Scale, 400 μm. Reproduced from [30]. (D) Astrocytes display robust [Ca²⁺]_i response, which propagates through the syncytium of confluent astrocytes after scratch injury, that is attenuated by TTX and KB-R7943. Modified from [30].

Astrocytic expression of VGSCs is highly dynamic, with expression depending on animal age and time in culture. For example, the proportion of hippocampal astrocytes expressing sodium currents, and channel densities, were found to decrease with time in culture, from ~75% (~0.6 channels/μm²) at day 1 to ~30% (with a much lower density) at later stages. The initial wave of VGSC expression was followed by a second wave of different VGSCs [22]. Astrocytes from different CNS regions exhibit different patterns of VGSC expression. VGSC expression is especially notable in spinal cord astrocytes where the mean Na⁺ channel density reaches ~2 channels/μm² and approaches 10 channels/μm² for some cells, 1-3 orders of magnitude larger than for astrocytes from other CNS regions [14, 15].

Astrocytic VGSC expression is influenced by multiple factors, including yet-unknown neuronal factors (for example, coculture with dorsal root ganglion neurons or exposure to neuron-conditioned media substantially reduces VGSC expression in spinal cord astrocytes) [23] and serum-borne agents [24]. Astrocytic expression of VGSCs can change markedly under pathological conditions. Reactive astrocytes in a rat model of epilepsy [25] and human astrocytes from epilepsy patients express increased levels of VGSCs [26, 27] with some studied specifically pointing to Nav1.5 [28]. Reactive astrocytes in and surrounding multiple sclerosis (MS) lesions, cerebral infarcts and brain tumors show marked upregulation of Nav1.5 (Figure 2A) [19]. The level of astrocytic Nav1.5 expression correlates with disease severity in a mouse model of MS [29].

Building on observations of Nav1.5 in reactive astrocytes in MS [19], Pappalardo et al [30] used siRNA to knock-down Nav1.5, as well as pharmacological tools, in an in-vitro model of glial injury (Figure 2C), and demonstrated that Na⁺ influx via Nav1.5 channels triggers reverse (Ca²⁺-importing) operation of the Na⁺/Ca²⁺ exchanger (NCX) within astrocytes, with resultant changes in intracellular Ca²⁺ that strongly modulate the glial response to injury (Figure 2C,D; Figure 3E). VGSC function in astrocytes has also been linked to the regulation of Na⁺/K⁺-ATPase activity, which is essential for maintaining ionic homeostasis in the CNS [31].

Figure 3. VGSCs in microglia.

(A) LPS-stimulated microglia (red) exhibit phagocytosis of fluorescent-labeled latex beads (yellow) which is markedly attenuated by incubation with 0.3 μm TTX. Modified from [44]. (B) Nonstimulated microglia exhibit a diffuse distribution of phalloidin (a marker for actin) and Nav1.6 immunolabeling (not shown), whereas ATP stimulation of microglia induces formation of lamellipodia that display robust immunolabeling for Nav1.6 (red) that co-localizes with phalloidin (green). Colocalization of phalloidin and Nav1.6 is shown in merged image (yellow). Scale, 10 μm. Modified from [45]. (C) Sodium channels contribute to lamellipodia formation in ATP-stimulated microglia. (Top panels). Nonstimulated (control) microglia display limited lamellipodia, as identified by phalloidin (green) accumulation. In contrast, ATP stimulation elicits lamellipodia formation in most microglia. Pretreatment with TTX (0.3 and 10 μM) substantially attenuates the formation of lamellipodia in ATP stimulated microglia. (Bottom panels). Images of top panels are displayed with labeling of phalloidin (green) and Iba1 (red), a marker of microglia, to clearly identify lamellipodia in microglia. Scale 5 μm. The graph below images represents quantification of lamellipodia formation. Reproduced from [45]. (D) (Left) Representative sodium current traces recorded from microglial cell before (black) and after (red) treatment of 0.3 μM TTX, and after wash-out (blue). (Right) Normalized peak current–voltage relationship of sodium current in microglia indicates the presence of two distinct populations of channels. Modified from [45]. (E) Schematic of putative pathway of sodium channel contribution to intracellular Ca²⁺ levels and downstream pathways. Depolarization of glial membrane leads to activation of VGSCs (Nav) allowing influx of Na⁺. Increased [Na⁺]_i induces reverse operation of the sodium-calcium exchanger (NCX), contributing to the level of [Ca²⁺]_i. Ca²⁺ signaling initiates downstream effects on cellular functions. Blockade of sodium channels with TTX , and of reverse NCX operation with KB-R7943, attenuates [Ca²⁺]_i levels. Modified from [30, 45].

Oligodendrocytes and Schwann Cells

Oligodendrocytes (OLs) and Schwann cells are myelin-forming cells of the CNS and PNS [32]. Oligodendrocyte precursor cells (OPCs) and NG2+ cells are immature precursors that can generate oligodendrocytes during development and following demyelination, and persist in the adult CNS [33].

Patch-clamp demonstration of multiple types of VGSCs in hippocampal NG2+ cells [34] is supported by RNA-Seq data, which shows mRNA for Nav1.3, Nav1.2, Nav1.8 and Nav1.1 [35]. [32, 36] identified Nav1.2 mRNA and protein in immature OLs in mouse and developing baboon brainstem and cerebellum and observed that these cells, under some conditions, exhibited Nav1.2-driven action potentials. Peak expression of VGSCs within OLs and their precursors coincides with the onset of myelination, [36] speculate that Nav1.2 expression is crucial for the spiking of immature oligodendrocytes which triggers their transitioning between progenitor cells and pre-myelinating OLs.

VGSCs are also present within Schwann cells, although less is known about their functional role. mRNA for Nav1.2 and Nav1.3 [37] and for Nav1.6 [20] have been reported. Early patch-clamp studies [38] suggested a channel density of about 2 channels/μm² with no apparent difference between myelinating and non-myelinating Schwann cells [39]. The functional role of VGSCs in Schwann cells is not yet fully understood.

Dendritic Cells and Macrophages

There is growing evidence for VGSC expression in dendritic cells (DCs) and macrophages, both originating from bone marrow. Expression of Nav1.7 is dynamic in human DCs, with expression being higher in immature DCs [40]. The frequency of cells expressing Na⁺ current and current density are significantly higher in the CD1a⁺ DC population than in CD1a⁻ cells, suggesting a role of Nav1.7 in DC differentiation and activation [40]. The expression of Nav1.7 in DCs and macrophages is regulated by cytokines and other inflammatory stimuli. Nav1.7 regulates the membrane potential and migration of CD1a+ human monocyte-derived DCs [40].

Nav1.5 and Nav1.6 proteins have been identified in human monocyte-derived macrophages where they are localized to intracellular organelles [41]. Nav1.5 is localized on the late endosome, where it regulates endosomal acidification and phagocytosis [41]. Nav1.6 protein is found on cytoplasmic vesicles associated with the actin cytoskeleton [41, 42]. Expression of Nav1.6 protein in rodent macrophages and microglia can be modulated by inflammatory stimuli such as cytokines, and is increased in experimental autoimmune encephalomyelitis (EAE), an animal model of MS [43]. Moreover, Nav1.6 channel regulates phagocytosis in mouse macrophages and microglia [43], consistent with the idea that Nav1.6 channel may play a role in the inflammatory response in the CNS.

Microglia

Microglia, the resident immune cells of the CNS, express multiple VGSCs. Microglia cultured from neonatal rat brains express Nav1.1, Nav1.5 and Nav1.6 proteins [44], with functional TTX-S currents confirmed by patch-clamp (Figure 3D) [45]. VGSC expression in microglia is developmentally regulated and is altered under pathological conditions. A robust increase in Nav1.6 expression in activated microglia and macrophages in EAE correlates with disease severity [43]. There is a significant increase in Nav1.6 expression in activated microglia in acute MS lesions [43].

The functional role of VGSCs in microglia is still under investigation, but evidence suggests they are involved in the regulation of multiple effector functions. In rat microglia, blockade of sodium channels with TTX and phenytoin significantly attenuates phagocytosis, migration, and release of pro-inflammatory cytokines [44]. Nav1.6 appears to play a very strong role in the regulation of phagocytic activity of rat microglia (Figure 3A) [43, 44]. Additionally, Nav1.6 channels accumulate in lamellipodia of activated microglia (Figure 3B,C), where they modulate Ca²⁺ transients and exert downstream effects on the activation of Rac1 and ERK1/2, regulatory proteins that play key roles in microglial migration [45]. It has been proposed that sodium influx through VGSCs leads to an increase in intracellular calcium levels, either through the reverse mode of the NCX or by depolarization-induced activation of voltage-gated calcium channels (VGCCs) (Figure 3E) [44, 45]. This increase in intracellular calcium can then trigger downstream signaling cascades.

Given the role of VGSCs in microglial activation and function, these channels may represent potential therapeutic targets for disorders characterized by neuroinflammation. Phenytoin, carbamazepine, and other sodium channel blockers are known to attenuate neuroinflammation and improve outcome in EAE [43, 44, 46-49], but more studies are needed.

Endothelial Cells

Endothelial cells (ECs) form the inner lining of blood vessels. mRNA and protein for Nav1.2 [50, 51], Nav1.5 [50], Nav1.6 [50, 51], and Nav1.7 [50, 52] have been detected in endothelial cells. Multiple studies have reported the presence of Na⁺ currents attributable to these channels in these cells including human endothelial cells [50, 53, 54].

The expression of VGSCs in ECs appears to be dependent on factors such as developmental stage, location within the vascular tree, and the presence of disease. VGSC expression in human saphenous vein endothelial cells (HSVECs) was found to be dependent on the age and disease state of the type of serum to which they are exposed, with serum from younger healthy donors significantly increasing VGSC expression when compared to serum from non-diabetic older patients with peripheral arterial disease [54], suggesting that the expression of VGSCs in ECs may be altered in certain disease states.

Several functional roles of VGSCs in ECs have been proposed, including the regulation of angiogenesis. VGSC activity has been shown to influence multiple angiogenic properties of human umbilical vein endothelial cells (HUVECs), including proliferation, chemotaxis, and tubular differentiation. These effects seem to be differentially controlled by VGSCs, with Nav1.7 primarily regulating chemotaxis and Nav1.5 influencing adhesion, tubulogenesis, and proliferation [50]. Another proposed function is the control of vasomotor tone, especially in the microcirculation, with Nav1.2, Nav1.6, and Nav1.9 potentially contributing to this response [51]. VGSC activity has also been found to modulate the endothelial cell response to shear stress [55].

Enterochromaffin Cells

Enterochromaffin Cells (EC) are enteroendocrine cells within the gastrointestinal tract that produce and release serotonin (5-HT) in response to various stimuli, thereby regulating gut motility, secretion, and sensation [56]. Recent studies have implicated EC cells in the pathogenesis of visceral pain and irritable bowel syndrome (IBS) [57-59]. A recent study [60] suggests Nav1.3 as the major VGSC subtype expressed in EC (Nav1.3 mRNA being the predominant subtype, and with voltage-clamp gating and pharmacological properties of functional channels consistent with the Nav1.3 subtype). This study showed that Nav1.3 channels can support spontaneous AP firing in EC cells with a role in regulating 5-HT release, consistent with earlier reports [61]. Interestingly, other studies [57] found that EC cells drive visceral pain and anxiety and were necessary for hypersensitivity in a mouse model of IBS. These findings suggest that the Nav1.3 channel may be a promising therapeutic target for the treatment of visceral pain and IBS [56-60].

Fibroblasts

Fibroblasts play a critical role in wound healing, tissue repair, and the production of collagen. The expression of VGSCs in fibroblasts appears to be context-dependent. Human atrial fibroblasts predominantly express Nav1.5 upon differentiating into myofibroblasts [62]. In contrast, human dermal fibroblasts express TTX-S Na⁺ channels in about 20% of cells [63]. Nav1.2, Nav1.3, Nav1.6, and Nav1.7 mRNA, and Na⁺ currents attributable to them, have been observed in human cardiac fibroblasts [64]. Interestingly, high levels of Nav1.7 mRNA and protein were detected in lung fibroblasts from both normal and fibrotic lungs [52]. Expression of VGSCs in fibroblasts can be developmentally regulated and altered under pathological conditions. Nav1.5 expression is absent in undifferentiated human fibroblasts but emerges upon differentiation into myofibroblasts [62]. This suggests a potential role for Nav1.5 in the fibrotic process.

Functional roles of VGSCs in fibroblasts are still under investigation. It has been suggested that persistent sodium current generated by Nav1.5 in human atrial myofibroblasts could lead to increased intracellular calcium that contributes to atrial fibrosis and fibrillation [62, 65]. The expression of Nav1.7 in lung fibroblasts, particularly in fibrotic lungs, suggests a potential role in pulmonary pathologies [52]. It has been suggested that these VGSCs might be targeted as potential strategies for managing atrial fibrillation [62] or pulmonary diseases [52], but more research is needed.

Pancreatic β-cells

Pancreatic β-cells are crucial for maintaining glucose homeostasis via secretion of insulin. This process is tightly regulated and involves a series of electrical events mediated by various ion channels, including VGSCs. In healthy β-cells, VGSCs contribute to the generation of action potentials, which are crucial for insulin secretion. The resulting increase in intracellular calcium triggers exocytosis of insulin granules [66, 67].

The expression of VGSCs in pancreatic β-cells has been confirmed at the mRNA and protein levels. Nav1.3, Nav1.6, and Nav1.7 have been found in human, rodent, and canine pancreatic β-cells [67-69]. Nav1.7 is the most abundant Na⁺ channel in mouse β-cells, contributing more than 85% of the Na⁺ current in these cells [69]. Alterations in VGSC expression have been reported under pathological conditions like diabetes. Nerve growth factor (NGF) has been found to increase sodium current and Nav1.3 mRNA levels in rat β-cells, indicating that VGSC expression can be modulated by growth factors [70].

The role of VGSCs in the pathogenesis of diabetes has been a subject of recent investigation. [71] proposed that gain-of-function mutations in Nav1.7 could chronically depolarize the β-cell membrane, leading to impaired insulin secretion and glucose intolerance. Furthermore, the SCN1B gene, encoding the β1 subunit of VGSCs, has been linked to glucose regulation. Studies have shown that the loss of the β1 subunit in mice, a major regulatory subunit expressed with Nav1.7, reduces glucose-stimulated insulin and glucagon secretion, resulting in severe hypoglycemia [72].

The potential therapeutic implications of targeting VGSCs in diabetes have also been explored. Studies have shown that carbamazepine, a sodium channel blocker, can exert protective effects on islet β-cells [73, 74]. Partial inhibition of VGSCs by glucose or low (0.1 μM and 0.2 μM) concentrations of TTX has been shown to enhance insulin synthesis in INS-1 cells, a rat insulinoma cell line. However, complete inhibition of VGSCs by high (1 μM) TTX concentrations can impair β-cell health and reduce insulin content [75], highlighting the delicate balance required in modulating VGSC activity for therapeutic purposes.

Osteoblasts

Embryonic osteoblasts in situ express mRNA for Nav1.2 [76]. Nav1.3, Nav1.4, Nav1.5, Nav1.6, and Nav1.7 mRNA were detected in the human osteosarcoma cell line MG-63 and primary human osteoblasts [77]. Nav2.1, Navβ1, and Navβ3 mRNAs have also been reported. [77, 78] with patch-clamp studies demonstrated the presence of functional channels.

The function of VGSCs in osteoblasts is not fully understood. [77] demonstrated that osteoblasts express channels at a density that allows the firing of action potentials under some conditions, suggesting a potential role in cellular signaling. Antiepileptic drugs (AEDs), particularly carbamazepine and phenytoin, can negatively impact bone health and are associated with increased risk of fractures [79, 80]. Further research is needed to elucidate the precise mechanisms involved and to explore the clinical implications of targeting VGSCs in osteoblasts, as may occur in patients taking AEDs.

Chondrocytes

Chondrocytes produce and maintain the collagen matrix that gives cartilage its structure and resilience. While chondrocytes are not considered excitable cells, there is growing evidence for the presence of VGSCs in these cells [8, 81, 82]. Despite a notably low density, there is recent evidence for a powerful role of VGSCs in regulating the secretome and pathobiology of these cells [8].The presence of VGSCs in chondrocytes has been confirmed at the mRNA, protein, and functional levels [8, 83, 84]. Several VGSC transcripts, including those for Nav1.2, Nav1.3, Nav1.4, Nav1.6, Nav1.7 and Nav1.9 have been identified in human chondrocytes [8, 83, 84]. The presence of Nav1.7 protein was recently demonstrated in human osteoarthritis (OA) chondrocytes [8]. The presence of functional Nav1.7 channels was established [8] by demonstrating the presence of sodium currents sensitive to ProTx-II (a Nav1.7-specific blocker) in human OA chondrocytes (Figure 4C). Nav1.7 expression can be induced by pro-inflammatory cytokines associated with OA, such as TNF-alpha and IL-1β and is upregulated in human OA cartilage compared to healthy cartilage [8].

Figure 4. Ablation of chondrocyte Nav1.7 protects against OA.

Nav1.7 genetic deletion in chondrocytes (A) or inhibition of Nav1.7 functional channels by selective Nav1.7 blocker PF-04856264 (B) substantially attenuate cartilage loss in surgically-induced destabilization-of-medial-meniscus (DMM) mouse osteoarthritis (OA) model. Shown are safranin O and Fast Green-stained sections of knee-joints of mice with the indicated genotype. Scale, 50 μm. (C) Functional expression of Nav1.7 in OA patient chondrocytes. (Left) Sodium currents in control (black solid line) and following block of Nav1.7 currents with ProTx-II (black dotted line), and the resulting subtracted trace showing the pure Nav1.7 current (ProTx-II-S current, blue line). (Right) TTX-S currents in chondrocytes. Sodium currents in control solution (black solid line) and in the presence of 1 μM TTX (black dotted line), and the trace of their difference (TTX-S current, red trace). (D) Model showing the role of Nav1.7 in the regulation of chondrocyte biology in OA, via its action on the NCX sodium-calcium exchanger and regulation of HSP70 and midkine release. Modified from [8].

VGSCs may contribute to maintaining the resting membrane potential and regulating intracellular ion homeostasis [8, 82, 85]. The upregulation of VGSC alpha subunits in chondrogenic progenitor cells (CPCs) derived from osteoarthritic cartilage suggests a role in chondrocyte differentiation and cartilage development [84]. A role for VGSCs in chondrocyte proliferation has also been suggested [86]. In addition, the sodium channel β1-subunit (SCN1B) has been consistently detected in chondrocytes, and it may modulate the expression and function of VGSCs [82, 87].

The growing evidence suggests that, despite their low density (0.10–0.15 channels/μm²; ~400 channels per cell) VGSCs play a powerful role in shaping the biology of chondrocytes. The expression of Nav1.7 in only a subpopulation of chondrocytes (17% of human chondrocytes from OA patients) [8] raises the question of how a low density of these channels, in only a small number of these cells, can have a substantial effect on joint biology. In this regard, it is notable that Nav1.7 blockade results in increased release of HSP70 and midkine from chondrocytes, triggered by increased intracellular Ca²⁺ levels in response to altered Na⁺/Ca²⁺ exchange; this change in the chondrocyte secretome, which results in pro-anabolic and anti-catabolic effects, may act in a paracrine manner to bias the entire chondrocyte population as well as other cells associated with arthritic joints, particularly activated synovial fibroblasts and infiltrating immune cells. The key role of Nav1.7 in regulating the chondrocyte secretome is illustrated in Figure 4D [8]. Consistent with this schema, carbamazepine, a sodium channel blocker currently in clinical use, prevents cartilage loss in multiple animal models of OA [8]. The use of VGSC blockers, such as carbamazepine and PF-04856264 (a Nav1.7-specific inhibitor) as a potential disease-modifying treatment for OA is supported by studies showing that these drugs or Nav1.7 genetic deletion can prevent cartilage loss in animal models of OA (Figure 4A,B) [8]. However, high doses of lidocaine, another VGSC inhibitor, may have toxic effects on cartilage [88], introducing a note of caution.

Cancer cells

A wealth of research has demonstrated expression or overexpression of various VGSC subunits in a range of cancers, suggesting their role in malignant behaviors, particularly migration and invasion. Notably, the specific α-subunit that is predominantly upregulated appears to vary depending on the type of cancer [89-91]. The expression of VGSCs in cancer cells is often abnormal, with some cancer cells expressing VGSCs that are not normally found in the corresponding healthy tissue. The expression of Nav1.5 and Nav1.7 VGSCs in cancer cells has been associated with increased metastatic potential and poor prognosis [89, 90].

Nav1.5 has been found in several types of cancer cells, including breast, colon, and lung cancer cells and its expression in certain cancer types has been associated with increased metastatic potential and poor prognosis [90]. For example, in breast cancer, the expression of neonatal splice variant of Nav1.5 (nNav1.5) is associated with metastasis and patient death [92, 93]. In colon cancer, Nav1.5 expression is also associated with increased metastatic potential [94]. It has been reported that inhibiting VGSC activity with TTX or siRNA silencing of the Nav1.5 neonatal splice variant can reduce invasiveness and migration of cancer cells in culture [94-96].

Nav1.7 has also been found to be expressed in several types of cancer cells, including prostate, endometrial, lung, pancreatic, ovarian, and glioblastoma cells [97, 98] where its expression is associated with increased metastatic potential and poor prognosis [89, 99-101]. In prostate cancer, Nav1.7 mRNA is strongly (about 20-fold) upregulated in prostate cancer tissue compared to non-cancer tissue [102]. The expression of Nav1.7 in prostate cancer cells is regulated by an activity-dependent positive feedback loop involving protein kinase A (PKA) [103]. Nav1.7 mRNA is 25-fold more abundant in endometrial cancer biopsies than in healthy tissue [99]. The authors also found that blocking Nav1.7 activity with the inhibitor PF-05089771, a selective Nav1.7 blocker, induced endometrial cancer cell apoptosis and reduced tumor growth and lymph node metastasis. Nav1.7 mRNA, protein and functional channels are expressed in non-small cell lung cancer cell lines, and functional expression of Nav1.7 is necessary for epidermal growth factor (EGF)-mediated invasion of these cancer cells [104]. Nav1.7 protein is also expressed in pancreatic cancer cell lines [97] and ovarian cancer cells [101]. The authors found that the use of the local anesthetic lidocaine, which blocks VGSCs, improved the outcomes of patients with ovarian cancer. The expression of Nav1.7 is thought to contribute to glioblastoma cell invasiveness; knocking-down Nav1.7 expression in patient-derived glioblastoma cells impaired cell viability and tumor growth in vitro, and extends the survival of glioblastoma-bearing mice [105]. These studies, in large part based on observations in culture and preclinical animal models, might be interpreted as suggesting potential therapeutic value of VGSC blockers in some types of cancers. However, an observational study on a large patient cohort suggested that exposure to VGSC-inhibiting anticonvulsants might be associated with reduced survival in breast, bowel and prostate cancer patients. In contrast, VGSC-inhibiting antiarrhythmic medications producing block of persistent sodium current have been reported to be associated with improved cancer-specific survival [106]. Moreover, ranolazine, alone or in combination with anti-cancer agents, has been suggested as a safe anti-metastatic therapy [107]. There is need for more study before any conclusions can be drawn about potential therapeutic implications for the particular VGSC blocker.

VGSCs in other cell types

The presence of VGSCs has also been reported in multiple other cell-types (Table 1), including retinal Muller glial cells (suggested roles in development, ionic homeostasis, gliotransmitter release) [108, 109]; adrenal chromaffin cells (modulation of catecholamine release) [110-112]; cochlear hair cells (transmission of auditory information to the central nervous system, shaping spontaneous activity before hearing onset) [113-116]; lymphocytes (control of intracellular Ca²⁺, resting membrane potential, invasiveness and metastatic behavior) [117-119]; neutrophils (adhesion, migration, chemotaxis) [120-124]; erythrocytes [125]; retinal pigment epithelial cells (cell-cell communication and phagocytosis of photoreceptor outer segments) [126-128]; and keratinocytes (cytokine and ATP release [129-132], suggesting a contribution of VGSCs to inflammatory skin diseases like rosacea and psoriasis [132], and to pain in complex regional pain syndrome (CRPS) and post-herpetic neuralgia (PHN)) [129, 132]. The presence of VGSCs has also been documented in mesenchymal stem cells (MSCs), with suggested roles in migration, differentiation and transition to cancer [133-136], and odontoblasts, with suggested roles in dentinogenesis, sensory transduction and dental pain [137-140].

Concluding remarks

VGSCs, once primarily a focus of research in neurons, muscle cells and cardiomyocytes where their role in action potential electrogenesis has been very well studied, are now known to be present and functional in many other cell types. The density of VGSCs in these non-excitable cells is in many cases very low, at least an order of magnitude lower than in excitable cells such as neurons, and this has made their study challenging. The dynamic nature of VGSC expression in these cells introduces another experimental challenge, since the pattern of VGSC expression can change depending on developmental stage, exposure to multiple factors, and under pathological conditions. Development of enhanced patch-clamp methods and refined culture protocols offers new experimental approaches, and RNAseq and development of IPSC models can offer additional insights.

An overarching principle is that, despite their low numbers, VGSCs in these cells can have substantial effects, even in the absence of production of all-or-none action potentials, on effector functions that include proliferation, migration, differentiation, secretome tuning, and immune responses. In some cases VGSCs influence cell function via a cascade in which Na⁺ influx biases Na⁺/Ca²⁺ exchange with resultant changes in Ca²⁺ levels. The development of ion-sensitive dyes, and the development of subtype-specific channel blockers, have provided important tools for this work. However, despite this recent progress, many questions remain unanswered (see Outstanding questions).

Our new understanding of VGSC activity in non-excitable cells may have implications for a number of diseases. For instance, the regulatory roles of VGSCs in immune cells like macrophages and microglia suggest potential roles in autoimmune diseases and neurodegenerative disorders. The possibility of modulating the glial scarring response in CNS disorders via targeting of astrocytic VGSCs deserves study. Recent studies have highlighted the strong role of VGSCs within chondrocytes in the pathogenesis of osteoarthritis, suggesting that it might be possible to protect joints from degeneration via the targeting of VGSCs. Developing selective VGSC blockers or modulators, including expression modulators, that can fine-tune channel activity in specific cell-types might offer novel treatment options for a wide range of diseases. Observations of dysregulated VGSC expression and of potential involvement in tumor growth, invasion, and metastasis, and of attenuation of metastatic behavior with VGSC block or knock-down in culture, have triggered early observational studies, but more study is needed.

The Hodgkin-Huxley model has delivered several generations of lessons about excitable cells such as neurons, muscle cells, and cardiomyocytes, whose major function is known to depend on action potential signaling supported by high densities of VGSCs. It is now clear that VGSCs are present and functional in multiple cell types that were traditionally not considered to be excitable, and that, despite their sparse expression, these channels are active participants in cellular processes with far-reaching implications for health and disease. Fortunately, discovery of VGSCs in these cells has been paralleled by increasing resolution of electrophysiological and optical methods. Continued research in this field promises to unravel the intricate mechanisms of VGSC function in these electrically quiet cell types and may unlock their therapeutic potential.

Box 1. Potential therapeutic implications of VGSCs in non-excitable cells.

While this article primarily focuses on the functional role of VGSCs in non-excitable cells, (dys)function of these channels has been implicated in many pathological conditions, some of them are listed below.

Multiple sclerosis (MS).

Reactive astrocytes in MS lesions upregulate Nav1.5, and this upregulation is linked to glial scarring, which can impede remyelination and axonal regeneration [19]. In animal models of MS, the expression of Nav1.6 is upregulated in activated microglia and macrophages, and blocking Nav1.6 reduces their phagocytic activity and the release of pro-inflammatory cytokines [43, 44]. Similarly, other sodium channel blockers, such as flecainide and safinamide, have shown promise in reducing microglial activation and protecting axons in models of MS [49].

Epilepsy.

Reactive astrocytes in epilepsy patients express increased levels of VGSCs, particularly Nav1.6. This upregulation may contribute to neuronal hyperexcitability and seizure generation [25].

Pain.

In inflammatory skin diseases, such as psoriasis, the increased expression of Nav1.8 in keratinocytes has been linked to the production of pro-inflammatory cytokines and inflammation [132]. It has been speculated that, in neuropathic pain disorders such as CRPS and PHN, altered VGSC expression in keratinocytes may contribute to the development and maintenance of pain [129-131].

Dermatological disorders.

There is an upregulation of Nav1.8 in keratinocytes, the predominant cell type in the epidermis, in rosacea and psoriasis. This upregulation is thought to contribute to the production of pro-inflammatory cytokines. Targeting Nav1.8 in keratinocytes has been shown to ameliorate skin inflammation in mouse models of rosacea and psoriasis [132].

Atrial fibrillation (AF).

A persistent sodium current generated by Nav1.5 in human atrial myofibroblasts has been suggested to contribute to atrial fibrosis and fibrillation [62].

Pulmonary Diseases.

The expression of Nav1.7 in lung fibroblasts, particularly in fibrotic lungs, suggests a potential role in pulmonary pathologies [52].

Ischemia-Reperfusion Injury.

Nav1.3 is expressed in neutrophils recruited to ischemic tissues and plays a role in neutrophil recruitment and function [120]. Modulating Nav1.3 activity may offer a potential therapeutic approach for ischemia-reperfusion injury [120, 121].

Osteoarthritis (OA).

Nav1.7 is upregulated in osteoarthritis cartilage and contributes to OA progression by affecting intracellular Ca²⁺ signaling and the chondrocyte secretome. Blocking or deletion of Nav1.7 within chondrocytes has shown promise in preventing cartilage loss in animal models of OA [8].

Cancer.

Nav1.5 is expressed in various cancer cells, including breast, colon, and lung cancer cells, and its expression in certain cancer types has been associated with increased metastatic potential and poor prognosis [90, 92, 93]. Nav1.7 is also expressed in several cancer types, including prostate, endometrial, lung, pancreatic, ovarian, and glioblastoma cells, and its expression is linked to increased metastatic potential and poor prognosis [89, 99-101]. VGSC blockers have shown promise in inhibiting cancer cell proliferation, migration, and invasion in preclinical studies [95, 101, 141]. However, some aspects of the preclinical data have been challenged by a large clinical study [106]. Ranolazine, alone or in combination with appropriate therapies, has been suggested as a safe anti-metastatic drug offering potential advantages over current systemic treatment modalities [107].