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Noncanonical Roles of Voltage-Gated Channels

Joel A. Black1,2 and Stephen G. Waxman1,2,* 1Department of , School of Medicine, New Haven, CT 06520, USA 2Center for Neuroscience and Regeneration Research, Veterans Affairs Connecticut Healthcare System, West Haven, CT 06516, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2013.09.012

The Hodgkin-Huxley formulation, at its 60th anniversary, remains a bastion of neuroscience. Sodium chan- nels Nav1.1–Nav1.3 and Nav1.6–Nav1.9 support electrogenesis in and are often considered ‘‘neuronal,’’ whereas Nav1.4 and Nav1.5 drive electrogenesis in skeletal and . These channels are, however, expressed in types that are not considered electrically excitable. Here, we discuss expression in diverse nonexcitable cell types, including astrocytes, NG2 cells, microglia, macro- phages, and cancer cells, and review evidence of noncanonical roles, including regulation of effector func- tions such as , motility, Na+/K+-ATPase activity, and metastatic activity. Armed with powerful + 2+ techniques for monitoring channel activity and for real-time assessment of [Na ]i and [Ca ]i, are poised to expand the understanding of noncanonical roles of sodium channels in healthy and diseased tissues.

In Brief able (termed here ‘‘nonexcitable cells’’). Moreover, there is abun- Sodium channels support electrogenesis in neurons and skeletal dant evidence demonstrating that sodium channels participate and cardiac muscle. They are, however, expressed in cell types in or regulate multiple effector functions in these nonexcitable that are not considered electrically excitable, including astro- cells. It is becoming clear, for example, that sodium chan- cytes, NG2 cells, microglia, , and cancer cells, nels—located not only on the plasma membrane delimiting the where they regulate phagocytosis, motility, Na+/K+-ATPase cell from the extracellular space but also, in some cases, on activity, and metastatic activity. intracellular membranes surrounding specific within We have now passed the 60th anniversary of the Hodgkin and the cell—contribute to processes as diverse as phagocytosis, Huxley (1952) discovery of the role of sodium channels in electro- motility, the release of bioactive molecules, and the regulation genesis, and it remains a bastion of modern neuroscience: of Na+/K+-ATPase activity in nonneuronal cells, including cells voltage-gated sodium channels are major players in action- as disparate as microglia and astrocytes within the CNS, where potential electrogenesis. Seven of the voltage-gated sodium they participate in the response to CNS injury, and cancer cells, channels (Nav1.1–Nav1.3 and Nav1.6–Nav1.9) play major where they contribute to motility and invasiveness. The neurosci- roles in electrogenesis in neurons and are often considered ence community, which has a long history of discoveries on ‘‘neuronal,’’ whereas Nav1.4 is the muscle sodium channel and sodium channels and their function and which possesses an Nav1.5 is the predominant cardiac myocyte channel. The canon- armamentarium of powerful tools that can help explicate the ical role of sodium channels in impulse electrogenesis and con- function of sodium channels, is in a unique position to elucidate duction in these excitable cells has been well established and is the functions of sodium channels in nonexcitable cells, as well as relatively well understood (see Rush et al., 2007; Catterall, 2012). in neurons. In this article, we discuss the expression of sodium Indeed, major aspects of the pathophysiology of neurological channels in nonexcitable cells and review accumulating evi- disorders, including (George, 2004; Helbig et al., dence showing that, within these cells, these channels play 2008; Reid et al., 2009; Eijkelkamp et al., 2012; Oliva et al., noncanonical roles and participate in multiple, diverse effector 2012), (MS) (Waxman, 2006), peripheral neu- functions. ropathy (Faber et al., 2012a,2012b), neuropathic (Wood, 2007; Dib-Hajj et al., 2010,2013), muscle diseases such as myo- Sodium Channels Are Expressed in Nonexcitable Cells tonic dystrophy (Jurkat-Rott et al., 2010; Stunnenberg et al., It is now known that nine different encode nine distinct 2010) and (Cannon 2010), and cardiac disor- sodium channels (Nav1.1–Nav1.9), which are expressed with ders such as (Campuzano et al., 2010; diverse temporal and regional patterns in excitable cells (Catter- Song and Shou, 2012; Tarradas et al., 2013), can be attributed all et al., 2005) and are variably associated with b-subunits to abnormalities of electrical excitability due to sodium channel (Patino and Isom, 2010; Brackenbury and Isom, 2011). Although dysfunction. Nonetheless, beyond the expression of sodium all voltage-gated sodium channels share a common overall channels in neurons, myocytes, cardiomyocytes, and other structural motif and considerable , the different sub- excitable cells, there is convincing evidence that multiple sub- types display distinct voltage dependence and kinetic and phar- types of voltage-gated sodium channels—the same channels macological properties (Catterall et al., 2005), and the repertoire that support electrogenesis in nerve cells, muscle cells, and car- of sodium channel subtypes expressed in a particular type of diac myocytes—are expressed and play major functional roles in excitable cell significantly contributes to its pattern of electrores- cell types that have been traditionally considered to be nonexcit- ponsiveness (see, e.g., Waxman 2000; Rush et al., 2007).

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Table 1. Voltage-Gated Sodium Channels in Nonexcitable Cells Cell type Sodium Currents or Channels References Astrocytes TTX-S, TTX-R currents; Nav1.2, Bevan et al., 1985, 1987; Nowak et al., 1987; Barres et al., 1988; Sontheimer Nav1.3, Nav1.5, Nav1.6 et al., 1991, 1992, 1994; Sontheimer and Waxman, 1992; Thio and Sontheimer, 1993; Black et al., 1995a, 1998, 2010; Kressin et al., 1995; Bordey and Sontheimer, 1998; Reese and Caldwell, 1999 Cancer cells Nav1.4, Nav1.5, Nav1.6, Nav1.7 Fraser et al., 2003, 2005; Roger et al., 2003; Diss et al., 2004, 2005; Brackenbury and Djamgoz, 2006; Gillet et al., 2009; Yang et al., 2012 Chrondrocytes TTX-S Sugimoto et al., 1996 Dendritic cells Nav1.7 Zsiros et al., 2009; Kis-Toth et al., 2011 Endothelial cells TTX-S, TTX-R currents; Nav1.4, Gordienko and Tsukahara, 1994; Gosling et al., 1998;Walsh et al., 1998; Nav1.5, Nav1.6 Traub et al., 1999 Fibroblasts TTX-S, TTX-R; Nav1.2, Nav1.3, Munson et al., 1979; Estacion 1991; Li et al., 2009; Chatelier et al., 2012 Nav1.5, Nav1.6, Nav1.7 Keratinocytes Nav1.1, Nav1.6, Nav1.8 Zhao et al., 2008 Islet b-cells TTX-S; Nav1.2, Nav1.3 Donatsch et al., 1977; Pressel and Misler, 1991; Philipson et al., 1993; Barnett et al., 1995; Eberhardson and Grapengiesser, 1999 Macrophages TTX-S current; Nav1.5, Nav1.6 Schmidtmayer et al., 1994; Carrithers et al., 2007, 2009, 2011; Black et al., 2013 Microglia TTX-S current; Nav1.1, Nav1.5, Korotzer and Cotman, 1992; No¨ renberg et al., 1994; Craner et al., 2005; Black Nav1.6 et al., 2009; Nicholson and Randall, 2009 Mu¨ ller TTX-S, TTX-R currents; Nav1.6, Chao et al., 1994; Francke et al., 1996; O’Brien et al., 2008; Linnertz et al., 2011 Nav1.9 Odontoblasts Nav1.2 Allard et al., 2006 Oligodendrocyte precursor TTX-S current Bevan et al., 1987; Williamson et al., 1997; Chen et al., 2008; Ka´ rado´ ttir et al., cells (NG2+) 2008; Tong et al., 2009 Osteoblasts Nav1.2 Black et al., 1995b Red blood cells Nav1.4, Nav1.7 Hoffman et al., 2004 Schwann cells TTX-S current; Nav1.2, Nav1.3 Chiu et al., 1984; Howe and Ritchie, 1990; Oh et al., 1994 T lymphocytes TTX-S, TTX-R currents; Nav1.5 DeCoursey et al., 1985, 1987; Lai et al., 2000; Fraser et al., 2004; Lo et al., 2012

Nav1.1, Nav1.2, and Nav1.6 are expressed in both central and glial and immune cells to endothelial and cancer cells, had peripheral neurons, whereas Nav1.7–Nav1.9 are preferentially been shown to express voltage-gated sodium currents, and it expressed in peripheral neurons (Beckh et al., 1989; Felts has been shown that nonexcitable cells can express multiple et al., 1997; Gong et al., 1999; Schaller and Caldwell, 2003; Cat- sodium channel subtypes (Table 1). terall et al., 2005; Dib-Hajj et al., 2013). Nav1.3 is present in the Patch-clamp recordings have confirmed the expression of adult human brain (Chen et al., 2000; Whitaker et al., 2001; Thim- functional sodium channels in cell types as divergent as astro- mapaya et al., 2005), but it is predominantly expressed during cytes (Barres et al., 1988; Sontheimer et al., 1992; Sontheimer embryonic and early postnatal periods in rodents. Nav1.3 is and Waxman, 1992), oligodendrocyte precursor cells (Son- upregulated in neurons in adult rodents after theimer et al., 1989; Kettenmann et al., 1991; Kressin et al., nerve injury (Waxman et al., 1994; Black et al., 1999) and contrib- 1995; Chen et al., 2008; Ka´ rado´ ttir et al., 2008), Schwann cells utes to hyperexcitability of these cells and thus neuropathic pain (Howe and Ritchie, 1990), fibroblasts (Estacion 1991; Li et al., (Cummins and Waxman, 1997; Samad et al., 2013). Nav1.4 is 2009), breast and prostate cancer cells (Fraser et al., predominantly expressed in (Barchi et al., 2003,2005; Roger et al., 2003; Brackenbury and Djamgoz, 1984; Trimmer et al., 1989), whereas Nav1.5 is the principal 2006; Ding et al., 2008; Gillet et al., 2009), and macrophages sodium channel in cardiac muscle (Gellens et al., 1992; Clancy and microglia (Korotzer and Cotman, 1992; No¨ renberg et al., and Kass 2002; Fozzard 2002). 1994; Schmidtmayer et al., 1994), including a microglial cell Early evidence of voltage-dependent sodium currents attribut- line derived from the human CNS (Nicholson and Randall, able to sodium channels in nonexcitable cells was provided by 2009). These recordings provide a precise measure of current patch-clamp studies on cultured Schwann cells and astrocytes density (and can thus be used for estimating the density of (Chiu et al., 1984; Bevan et al., 1985), which, when held functional channels in the ) and can distinguish at À100 mV and then stepped to +40 mV in 10 mV steps, ex- the expression of sodium channels that are sensitive to hibited a family of fast-activating, fast-inactivating traces that nanomolar levels of TTX (TTX-S; Nav1.1–Nav1.4, Nav1.6, and could be blocked with the sodium-channel-specific ligands Nav1.7) from that of a group of TTX-resistant (TTX-R) channels, (TTX) and (STX). Subsequent to these which require micromolar concentrations of TTX for blockade early studies, a wide variety of nonexcitable cells, ranging from (Nav1.5, Nav1.8, and Nav1.9). RT-PCR, in situ hybridization,

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and immunocytochemical techniques have provided evidence of 2009). OPCs express robust voltage-sensitive sodium currents. the molecular identities of sodium channel a-subunits in nonex- In contrast, sodium currents are smaller and present in only a citable cells and have shown the expression of every sodium subpopulation of more differentiated O4+ cells and are absent channel subtype in some type of nonexcitable cell (e.g., Black in mature oligodendrocytes (Sontheimer et al., 1989). The et al., 1998; Diss et al., 1998; Zhao et al., 2008; Zsiros et al., expression of sodium channels in OPCs may have important 2009). Within some cell types, only a single subtype has been physiological consequences. It is known that detected (e.g., Nav1.7 in dendritic cells; Zsiros et al., 2009), activity can to vesicular glutamate release at discrete sites whereas other cell types have been shown to express multiple along , and this can activate ionotropic glutamate recep- subtypes; for instance, astrocytes have been shown to express tors on neighboring OPCs (Kukley et al., 2007). Eighty-one Nav1.2, Nav1.3 (Black et al., 1995a), Nav1.5 (Black et al., percent of OPCs that express sodium channels respond to this 1998), and Nav1.6 (Reese and Caldwell, 1999), and microglia type of release, whereas only 2% of OPCs have been shown to express Nav1.5 and Nav1.6 (Craner et al., that lack sodium channel expression show these responses, 2005; Black et al., 2009). Human red blood cells have been suggesting that the ability of OPCs to respond to axonal shown to express Nav1.4 and Nav1.7 (Hoffman et al., 2004). signaling may be associated with the expression of sodium Although this article focuses on the Nav1.1–Nav1.9 pore-forming channels within these cells (Ka´ rado´ ttir et al., 2008). In fact, it a-subunits of sodium channels, there is also evidence demon- has been hypothesized that the activity of these channels may strating the presence of sodium channel b-subunits in some trigger myelination by these cells (Ka´ rado´ ttir et al., 2008). One nonexcitable cells (Oh and Waxman, 1994), where they are possible mechanism is that expression of sodium channels in postulated to function as cell-adhesion molecules (Brackenbury OPCs participates in setting their resting , et al., 2010). and this, in turn, may contribute to controlling their rate of prolif- As described below, voltage-gated sodium channels con- eration (Xie et al., 2007). tribute to diverse effector functions of nonexcitable cells. This Switches in sodium current expression are not confined to is all the more remarkable because, in general, estimated den- healthy nonexcitable cells but have also been reported after sities of sodium channels in nonexcitable cells are substantially pathological challenge. For example, in an in vitro model of lower than those in excitable cells (<1 versus 2–75 mmÀ2, respec- astrogliosis in which a confluent layer of cultured rat astrocytes tively; see, e.g., Sontheimer et al., 1992). Spinal cord astrocytes was scratched for the production of a linear injury, the injury in vitro are an exception and can express sodium channels at evoked a switch from TTX-S currents to TTX-R sodium currents densities estimated to be as high as 10 channels per mm2 (MacFarlane and Sontheimer, 1998). Consistent with the change (Sontheimer et al., 1992), which, although not supporting action in sodium currents, markedly upregulated expression of the potential electrogenesis close to , can support TTX-R sodium channel, Nav1.5, was recently reported in reac- the production of all-or-none overshooting action-potential-like tive astrocytes at the boundary of the scar injury in this in vitro responses when the cells are hyperpolarized to levels that model (Samad et al., 2013). Importantly, upregulation of sodium remove resting inactivation (Sontheimer and Waxman, 1992). channels in reactive astrocytes occurs in situ within the human The density of sodium channels in cells such as astrocytes brain. Observations on rapid-autopsy tissues demonstrate in vitro depends on the milieu to which the cells are exposed robust upregulation of Nav1.5, which is not seen in normal con- (Thio and Sontheimer, 1993; Thio et al., 1993), raising the ques- trol brains, within scarring astrocytes in acute and chronic MS tion of whether channel expression is an artifact of culture. plaques and adjacent to cerebrovascular accidents and neo- Importantly, however, astrocytes within slices of hippocampus, plastic lesions in the human brain (Black et al., 2010; Figure 1). cerebral cortex, spinal cord, and cerebellum also express Astrocytes are not the sole nonexcitable cell type that displays sodium currents (Sontheimer and Waxman, 1993; Chva´ tal a switch in sodium channel expression after pathological insult. et al., 1995; Bordey and Sontheimer, 2000). Thus, expression The differentiation of human fibroblasts into myofibroblasts of these channels within astrocytes can occur within a relatively under pathological conditions is accompanied by de novo normal milieu and is not an artifact of culture. Further confirma- Nav1.5 expression (Chatelier et al., 2012) similar to that exhibited tion of this comes from immunocytochemical studies that have by reactive astrocytes. Mu¨ ller cells, the primary macroglial cells demonstrated the expression of sodium channels in astrocytes in the , where they provide functional and metabolic sup- in situ within both the rodent (Black et al., 1994, 1998) and the port to neighboring neurons (Bringmann and Wiedemann, human (Black et al., 2010) brain. 2012), also exhibit increased sodium channel expression in response to injury (e.g., glaucoma, melanoma, retinal detach- Sodium Channel Expression in Nonexcitable Cells Is ment) in that they express 4-fold larger sodium currents than Dynamic do Mu¨ ller cells from normal (12.2 versus 3.0 pA/pF, The expression of sodium channels in nonexcitable cells is not respectively; Francke et al., 1996). static and, on the contrary, may change markedly depending on the developmental and/or physiological state of the cells. Sodium Channels Participate in Multiple Effector For instance, differentiation of cells of the oligodendrocyte line- Functions of Nonexcitable Cells age, from oligodendrocyte precursor cells (OPCs) to mature Several lines of evidence have demonstrated that sodium chan- myelinating oligodendrocytes, is accompanied by switches in nels contribute to multiple, diverse cellular functions in nonexcit- patterns of phenotypic expression (see Levine et al., 2001), able cells (Table 2). In many cases, a functional role has including the downregulation of sodium channels (Paez et al., been demonstrated on the basis of pharmacological blockade,

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Figure 1. Nav1.5 Expression in Astrocytes within MS Tissue A montage of gray-scale images of R. communis agglutinin I (RCA-1)-positive macrophages (white) in a section of MS tissue was constructed to provide orientation for localization of astrocytes. A region of substantial infiltration is present in the lower three-quarters of the field. An adjacent serial section was labeled for Nav1.5 (red), GFAP (green), and PLP (blue), and high- magnification images corresponding to the boxed numbers in the montage are shown in the right- hand panels. A GFAP-positive astrocyte within normal-appearing white matter (NAWM), which is not in the montage field, exhibited limited Nav1.5 immunolabeling. In contrast, an astrocyte (panel 1) within white matter, which is more disrupted than NAWM, and 250 mm from the plaque border was hypertrophic and showed an upregulation of Nav1.5. Astrocytes at the border (panel 2) and within (panel 3) the active lesion displayed intense Nav1.5 immunolabeling. Colocalization of GFAP and Nav1.5 is shown in yellow. (Modified from Figure 7 in Black et al., 2010).

phage plasma membrane. Evidence of a role for Nav1.5 in macrophage function was provided by the demonstration that TTX at 10 mM (a concentration that blocks Nav1.5) and knockdown of Nav1.5 with small hairpin RNA (shRNA) inhibit phagocytosis by these cells. Using time- resolved fluorometry, Carrithers et al. knockout, or knockdown. For example, Craner et al. (2005) (2007) also demonstrated that the sodium channel activator demonstrated that sodium channel blockade with TTX attenu- veratridine reduces intraendosomal pH and intraendosomal ates phagocytosis by 40% in cultured microglia. They also [Na+] and that 10 mM TTX blocks lipopolysaccharide-induced showed that the phagocytic capacity of microglia derived from acidification of the intraendosomal compartment in both purified med mice, which lack Nav1.6 channels (Kohrman et al., 1996), endosomes and intact macrophages. These results suggest a is 65% lower than that of microglia from wild-type (WT) mice. model in which Nav1.5 channels, located intracellularly within Black et al. (2009) observed that the clinically used sodium chan- the membrane of late endosomes, provide a route for Na efflux, nel blocker significantly reduces the phagocytic which counterbalances proton influx, and thereby maintain elec- activity of microglia by 50%–60% (Figure 2). These studies troneutrality during acidification, which is one of the final stages also showed that TTX and phenytoin attenuate the release of of phagocytosis. Noting that pH regulates the current of Nav1.5 the proinflammatory interleukin 1-a (IL-1a), IL-1b, (Khan et al., 2006), Carrithers et al. (2007) suggested that the and tumor necrosis factor a (TNF-a) from stimulated microglia expression and activity of Nav1.5 on the intracellular endosomal while having minimal effects on the release of IL-2, IL-4, IL-6, membrane may provide a regulatory loop in which, as intraendo- IL-10, monocyte chemotactic 1 (MCP-1), and transform- somal pH decreases toward a functional optimum in the late ing growth factor a (TGF-a). Phenytoin and TTX also significantly endosome, the endosomal sodium channels become inactive, decrease ATP-induced migration of microglia. Supporting a role limit further acidification, and thus prevent cell injury. Extending for Nav1.6 in the pathway leading to microglial migration, the these observations to human tissue, Black et al. (2013) recently level of ATP-induced migration of microglia cultured from med demonstrated the expression of Nav1.5 in late, but not early, mice is significantly lower than that of cells from littermate WT endosomes in phagocytosing macrophages within acute MS mice (Black et al., 2009). lesions. Another line of evidence of a novel contribution of sodium Still another example of a role for sodium channels in the regu- channels—in this case the ‘‘cardiac’’ Nav1.5 channel—to the lation of effector functions of nonexcitable cells is provided by function of nonexcitable cells was provided by Carrithers et al. dendritic cells (DCs), the antigen-presenting immune cells that (2007), who demonstrated the expression of Nav1.5 within phag- function as intermediaries between innate and adaptive immu- osomes of activated human macrophages by immunocyto- nity. Immature monocyte-derived DCs express Nav1.7 chan- chemistry and immunogold electron microscopy (Figure 3). The nels, but the expression of these channels is attenuated during expression of Nav1.5 was restricted to late endosomes of these maturation (Zsiros et al., 2009). Even if it did not result in a cells and was not detected in early endosomes or on the macro- large increase in intracellular [Na+], the addition of even a small

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Table 2. Sodium Channel Contributions to Effector Functions Cell Type Sodium Channel Blockade or Activation References Astrocytes TTX: attenuates Na+/K+-ATPase activity and increases Sontheimer et al., 1994 cell death Cancer cells TTX, phenytoin, Nav1.5 siRNA: attenuate motility, Fraser et al., 2003, 2005; Roger et al., 2003; Brackenbury migration, invasiveness, metastasis, and extracellular et al., 2007; Brackenbury and Djamgoz, 2006; Gillet et al., acidification 2009; Yang et al., 2012; Yildirim et al., 2012 Dendritic cells (CD1a+) TTX, Nav1.7 siRNA: inhibit cell migration and sensitize Kis-Toth et al., 2011 cells for activation Endothelial cells TTX: potentiates shear-stress-induced ERK1/2 Traub et al., 1999 activation Islet b-cells TTX: attenuates release Donatsch et al., 1977; Pressel and Misler, 1991 veratridine: increases insulin release Keratinocytes TTX: inhibits ATP release Zhao et al., 2008 Macrophages TTX, Nav1.6 shRNA: attenuate phagocytosis and Carrithers et al., 2007, 2009 invasion and inhibit podosome formation Nav1.5 siRNA: attenuates endosomal acidification veratridine: increases endosomal acidification Microglia TTX, phenytoin: attenuate phagocytosis, migration, Craner et al., 2005; Black et al., 2009 release Oligodendrocyte precursor TTX, Nav1.x siRNA: attenuate -induced migration Tong et al., 2009 cells (NG2+) Retinal Mu¨ ller cells TTX, STX, phenytoin: inhibit ligand-induced release Linnertz et al., 2011 of glutamate T lymphocytes TTX: inhibits migration Fraser et al., 2004 standing sodium conductance (due, for example, to persistent potentiate the effect of epidermal growth factor (EGF), which activation of a small fraction of available channels) would be enhances the migration and invasiveness of prostate cancer expected to have a depolarizing effect on resting potential of cells (Uysal-Onganer and Djamgoz, 2007), suggesting a potential these cells. A link between Nav1.7 expression and cell function mechanism linking sodium channel activity to metastatic activity in DCs is suggested by the observation that Nav1.7 current in these cells. In this regard, it has been demonstrated that density is significantly greater in CD1a+ immature DCs than in sodium channel activity in MDA-MB-231 breast cancer cells CD1aÀ immature DCs (Kis-Toth et al., 2011). The presence of a to intracellular alkalinization and pericellular acidification, Nav1.7 current is associated with a depolarized resting mem- which promotes the activity of released proteolytic enzymes brane potential, as predicted by the Goldman-Hodgkin-Katz and cell invasiveness (Gillet et al., 2009). At present, the molec- equation, in CD1a+ DCs, and blockade or knockdown of ular pathway(s) by which sodium channel activity participates in Nav1.7 in these cells yields a hyperpolarization of the resting the regulation of intracellular and extracellular pH of cancer cells membrane potential, which is associated with attenuated is not known, but it has been suggested to involve interactions mobility and gene expression of matrix metalloproteinase 12 with the sodium-proton exchanger, bicarbonate transporters, (Kis-Toth et al., 2011). Nav1.7 blockade with TTX also attenuates vacuolar H+-ATPase, and/or monocarboxylate transporters (Gil- inflammatory-cytokine-stimulated activation of CD1a+ DCs and let et al., 2009). Sodium channel activity is also likely to affect the release of TNF-a and IL-10 from these cells. cancer cells’ membrane potential, which has been linked to their There is also increasing evidence suggesting that levels of proliferation and migration (Yang and Brackenbury, 2013). In sodium channel expression are correlated with invasiveness in vitro assays of cancer cell invasiveness, blockade, or knock- and metastatic potential in several types of cancer cells. down of sodium channels with TTX, phenytoin or small inter- For example, strongly metastatic breast cancer cells express fering RNA (siRNA) has been shown to significantly attenuate voltage-sensitive sodium currents, whereas weakly metastatic directional motility and cell migration (Roger et al., 2003; Fraser and normal breast epithelial cells do not (Fraser et al., 2005; et al., 2005; Brackenbury et al., 2007; Brackenbury, 2012; Yang et al., 2012). Upregulated expression of a neonatal splice Yang et al., 2012). At the in vivo level, it has been reported in a form of Nav1.5 has been linked to strong metastatic potential rat model of prostate cancer that blocking sodium channel activ- in vitro and breast cancer progression, and there appears to ity with TTX reduces the number of lung metastases by 40% and be a correlation between Nav1.5 expression and clinically significantly prolongs lifespan (Yildirim et al., 2012). Whether assessed lymph node invasion (Fraser et al., 2005). Levels of targeted sodium channel blockade or knockdown can contribute sodium channel expression are also greater in prostate biopsies to cancer treatment is not yet known, but higher levels in more from patients with cancer than in non-cancer biopsies (Diss metastatic cells suggest these channels as potential biomarkers et al., 2005). Sodium channel activity has been reported to (Diss et al., 2005).

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Figure 2. Sodium Channel Blockade Inhibits Microglial Phagocytosis Lipopolysaccharide (LPS)-stimulated microglia (red) in vitro exhibited substantial phagocytosis of fluorescent-labeled latex beads. Incubation with tetrodotoxin (TTX) and phenytoin (phen) attenuated phagocytosis of latex beads by LPS-activated microglia. (Modified from Figure 2A in Black et al., 2009).

Pathways of Sodium Channel Activity Regulating Cell these cells with an ability to respond appropriately to positively Function selecting ligands, to which these cells do not normally respond Although it is clear that sodium channels participate in or regu- (Lo et al., 2012). Sodium channel activity thus appears to late multiple functions within a spectrum of nonexcitable cell contribute to Ca2+ influx after stimulation in these T cells and types, we presently have only hints about the underlying molec- thereby play a critical role in positive selection after challenge ular mechanism(s). Extrapolating from the small number of cell by weak-signal ligands. How the Nav1.5 channels function within types in which mechanisms linking sodium channels to cell func- these cells to trigger calcium signals is not yet known. tion have been studied, it seems likely, in fact, that sodium chan- A role for sodium channels as a driver of reverse (Ca2+-import- nels affect physiological responses of cells by multiple pathways ing) Na/Ca exchange, which has been observed in multiple (Figure 4). In a study on astrocytes, Sontheimer et al. (1994) sug- nonexcitable cell types, including NG2 cells and astrocytes (Kir- gested a mechanism in which sodium channels provide a route ischuk et al., 1997; Paluzzi et al., 2007; Tong et al., 2009), is for a small sustained Na+ influx, which is necessary for continued beginning to emerge as a common motif. The Na/Ca exchanger operation of Na+/K+-ATPase. In these cells, sodium channel can operate in forward mode by carrying Na+ down their blockade with TTX attenuates Na+/K+-ATPase activity, reduces concentration gradient into cells and in return exporting Ca2+ + astrocyte [Na ]I, and with prolonged exposure, leads to cell or, if the cell is depolarized or the transmembrane gradient of death. This cascade, in which voltage-gated channels provide Na+ is reduced, can function in reverse mode by carrying Na+ a route for Na+ influx, which is required for maintaining ions out of the cell while importing Ca2+ (Annunziato et al., + [Na ]i at concentrations necessary for activity of the sodium 2004). In NG2 cells, which are sometimes referred to as oligo- pump, suggests that the flux of Na+ ions through sodium chan- dendrocyte precursors cells, Tong et al. (2009) demonstrated nels may provide a feedback loop that, in turn, modulates the increased intracellular Na+ and Ca2+ levels, coincident with ability of astrocytes to regulate K+ levels in the extracellular membrane and enhanced migratory capacity of space. It is not yet known whether sodium channels contribute the cells, that could be evoked by the application of GABA. to the regulation of Na+/K+-ATPase activity in a similar manner Blockade or knockdown of sodium channels by siRNA signifi- 2+ + in other cell types. cantly decreased the rise in [Ca ]i and [Na ]i, and attenuated Observations on T lymphocytes suggest that sodium channel the migration of NG2 cells (Tong et al., 2009). Attenuated 2+ activity can trigger calcium signals, which, in turn, fine-tune [Ca ]i and reduced cell migration were also observed after the effector functions of these cells. Sustained Ca2+ influx siRNA knockdown of the Na/Ca exchanger or KB-R7943 has been shown to be required for the positive selection of blockade of reverse Na/Ca exchange. Thus, within NG2 cells, CD4+CD8+ T cells (Oh-hora, 2009). Recent work has demon- Na+ flux through sodium channels, in this case triggered by strated the presence of Nav1.5 sodium channels in these cells GABA, elicits reverse operation of the Na/Ca exchanger and 2+ 2+ and has shown that blockade of these sodium channels with influx of Ca , resulting in increased [Ca ]i affecting cellular TTX, or knockdown with shRNA, inhibits the induction of sus- motility. A similar mechanism may operate in astrocytes. tained Ca2+ influx induced in CD4+CD8+ thymocytes by the posi- Although astrocytes do not generate action potentials under tively selecting ligand (gp250-I-EK) and prevents the positive physiological conditions and have thus been considered to be selection of CD4+ T cells (Lo et al., 2012). Moreover, a gain-of- electrically inexcitable, Verkhratsky and Kettenmann (1996)— function assay showed that ectopic expression of Nav1.5 chan- noting that these cells can produce transient Ca2+ signals, which nels in T cells obtained from mice with the transgenic expression can propagate as waves—have used the term ‘‘Ca2+ excitability’’ of a specific to moth cytochrome c bound to major his- to describe this phenomenon. Both forward and reverse opera- tocompatibility complex (MHC) class II molecule I-EK (Lo et al., tion of the Na/Ca exchanger have been described in astrocytes 2009) (these mice do not normally express Nav1.5) endows (Kirischuk et al., 1997; Paluzzi et al., 2007), which, as a result of

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Figure 3. Nav1.5 Colocalizes with Phagocytosed E. coli in Activated Macrophages Sixty minutes after bacterial challenge, Nav1.5 (green) and Texas-Red-labeled E. coli were colocalized in a perinuclear region. The nucleus was stained with DAPI (blue). (Modified from Figure 6B in Carrithers et al., 2007). their membrane potential and intracellular level of Na+, are detected in multiple nonexcitable cells, including astrocytes poised close to the of the Na/Ca exchanger, (Oh and Waxman, 1998), human macrophages (Carrithers which can therefore produce rapid, short-lived, and spatially et al., 2009), and cancer cells (Fraser et al., 2005; Brackenbury restricted Ca2+ signals (Kirischuk et al., 2012). The possibility et al., 2007). Although the functional consequences of expres- that activity of sodium channels can elicit reverse Na/Ca sion of these splice variants is not fully understood, it is known exchange and modify cellular responses in astrocytes, and in that expression of the Nav1.5 neonatal splice variant potentiates other cell types, is currently being examined. the invasiveness of breast cancer cells (Brackenbury et al., A different role for sodium channels in the modulation of cell 2007). Interestingly, alternative splicing of some sodium chan- motility is suggested by the intracellular localization of Nav1.6 nels, such as Nav1.6, can produce truncated and presumably near F-actin bundles in macrophages and melanoma cells in nonconducting two-domain , which are present in a areas of cell attachment, where a Nav1.6 splice variant regulates broad range of nonneuronal tissues (Plummer et al., 1997; Oh cellular invasion via modulation of the formation of podosomes and Waxman, 1998). It can also be speculated that further study (specialized F-actin zones, which mediate adhesion, invasion, will uncover nonconducting roles for sodium channels, as has and migration) and invadopodia (Carrithers et al., 2009). Sodium been proposed for the autoregulation of transcription by the channel blockade with 0.3 mM TTX and Nav1.6 knockdown with C terminus of the L-type Cav1.2 (Dolmetsch shRNA inhibit podosome formation and invasion through the et al., 2001; Gomez-Ospina et al., 2006; Satin et al., 2011) and basement membrane matrix. Further implicating Nav1.6, podo- tumor progression by the channel ether-a´ -go-go some formation was also attenuated in macrophages obtained (Kv10.1) (Downie et al., 2008). from med mice. Activation of sodium channels with veratridine triggered a shift of Na+ from cationic vesicular compartments Perspectives: An Opportunity for Neuroscientists to mitochondria and a rise in intracellular Ca2+ in macrophages, It is now clear that many cell types traditionally considered non- and blockade of the mitochondrial Na/Ca exchanger significantly excitable express voltage-gated sodium channels. Moreover, 2+ reduced the veratridine-induced increase in [Ca ]I within wild- there is abundant evidence that blockade or knockdown of type macrophages, but not in macrophages from med mice. sodium channel activity can significantly alter effector functions Taken together, these observations suggest that Nav1.6 and physiological responses of these nonexcitable cells. We do contributes—via a mechanism involving release of sodium not, at this time, have a full appreciation of the intracellular cas- from vesicular intracellular stores, uptake by mitochondria, and cades by which sodium channel activity contributes to signaling extrusion of Ca2+ from mitochondria—to the control of podo- pathways in nonexcitable cells. Data from recent studies sug- some and invadopodia formation and thereby regulates F-actin gest that there are multiple intracellular molecular mechanisms cytoskeletal remodeling and movement of macrophages and and provide hints that sodium channel activity in some cells melanoma cells. may amplify, localize, and/or fine-tune intracellular Ca2+ levels. An added layer of complexity may arise from the fact that These studies also indicate that, in at least some cell types, the sodium channels possess alternative splicing sites, many of activity of sodium channels localized within intracellular com- which are evolutionarily conserved and probably functionally partments, and not solely on the plasma membrane, can partic- important (e.g., Plummer et al., 1997; Diss et al., 2004; Gazina ipate in regulation of cellular functions. et al., 2010; Schroeter et al., 2010). The splice variants can Contributions of voltage-gated sodium channels to the func- have distinct biophysical properties, which in some cases are tion of nonexcitable cells should not be a surprise to neuro- dependent on interactions with b-subunits (Farmer et al., scientists. As a result of their voltage dependence and 2012). Neonatal splice variants of sodium channels have been kinetics, several sodium channels, notably Nav1.9 (Cummins

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Figure 4. Schematic Showing Multiple Modes of Participation of Sodium Channels in Effector Functions of Nonexcitable Cells (A) Nav1.5 expression within late endosomes of macrophages contributed to the regulation of acidification of endosomal lumina by counter- balancing proton accumulation, thereby main- taining electroneutrality. Endosomal sodium channels became increasingly inactive with acid- ification, providing a negative-feedback loop and preventing cell injury. (B) Expression and activity of sodium channels in nonexcitable cells can lead to depolarized resting membrane potential and modulation of multiple effector functions. (C) Expression of sodium channels in nonexcitable astrocytes provides a route for Na+ influx, which + maintains [Na ]i at levels necessary for the activity of the Na+/K+-ATPase. (D) Activity of sodium channels in nonexcitable + + cells results in Na influx and increased [Na ]I, leading to reverse operation of the Na/Ca exchanger and import of Ca2+ and modulation of .

but their precise contributions to the functions of these, as well as other, cell types in disease remains to be eluci- dated. Finally, given that sodium chan- nels are emerging as functional players in nonexcitable cells in disease states, we need to understand whether targeted blockade or knockdown of sodium chan- nel subtypes in specific cell types might be of therapeutic value. et al., 1999) and Nav1.7 (Cummins et al., 1998), participate in Neuroscientists, armed with an array of methods for directly electrogenesis in the subthreshold range, where they amplify monitoring channel activity by using electrophysiological, imag- small so as to bring the cell to the action- ing, or pharmacological techniques and with assays that permit potential threshold where other sodium channel subtypes real-time assessment of intracellular [Na+] and [Ca2+], are in a activate to produce the majority of the inward current respon- unique position to further elucidate the noncanonical roles of sible for the depolarizing phase of the action potential. voltage-gated sodium channels in cells that have traditionally Within the injured , persistent sodium currents, been considered nonexcitable. Many of these cell types interact, even in resting axons, can trigger injurious Ca2+-importing directly or indirectly, with neurons. We predict that over the next reverse Na/Ca exchange (Stys et al., 1992, 1993). However, in decade, neuroscientists will use tools already at their disposal to contrast to the roles played by sodium channels in the expand our understanding of the ensemble of sodium-channel- subthreshold domain in excitable cells (see Rush et al., 2007 mediated mechanisms that contribute to the function of normal for a review), the noncanonical roles of sodium channels, in and injured cells. cell types traditionally viewed as nonexcitable, have been rela- tively unexplored. Table 3. Noncanonical Roles of Voltage-Gated Sodium Channels: There is a world of sodium channel activity, within cells tradi- Unanswered Questions tionally viewed as nonexcitable, that has been ‘‘below the sur- What is the full array of cell types in which voltage-gated sodium face’’ to neuroscientists. Some of the unanswered questions channels are expressed? about the noncanonical roles of sodium channels are summa- What is the spectrum of effector functions regulated by sodium rized in Table 3. We need to know the full array of cell types in channels? which sodium channels are expressed and the full spectrum How do sodium channels accomplish their regulatory roles in of the functional roles of sodium channels in these cells. There nonexcitable cells? is a pressing need to understand the mechanisms by which How do sodium channels contribute to disease pathophysiology of sodium channels exert their influences in these cells. It is clear, nonexcitable cells? from the roles of sodium channels in phagocytosis and their up- Can sodium channels in nonexcitable cells be therapeutically regulation in glial cells in the injured nervous system, that these targeted? channels are poised to contribute to disease pathophysiology,

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