Multiple sodium channels and their roles in electrogenesis within dorsal root ganglion neurons

Abstract

Dorsal root ganglion neurons express an array of sodium channel isoforms allowing precise control of excitability. An increasing body of literature indicates that regulation of firing behaviour in these cells is linked to their patterns of expression of specific sodium channel isoforms, which have been discovered to possess distinct biophysical characteristics. The pattern of expression of sodium channels differs in different subclasses of DRG neurons and is not fixed but, on the contrary, changes in response to a variety of disease insults. Moreover, modulation of channels by their environment has been found to play an important role in the response of these neurons to stimuli. In this review we illustrate how excitability can be finely tuned to provide contrasting firing templates in different subclasses of DRG neurons by selective deployment of various sodium channel isoforms, by plasticity of expression of these proteins, and by interactions of these sodium channel isoforms with each other and with other modulatory molecules.

Introduction

Although electrogenesis in neurons has classically been considered to be the product of activity of ‘the’ sodium channel, we now know that multiple isoforms of voltage-gated sodium channels (Na_v) are present within neurons (Catterall et al. 2005). It is becoming increasingly clear that these multiple sodium channel isoforms collaborate in the production of electrical activity by neurons. Particularly fruitful for the study of sodium channels and their roles in electrogenesis are the neurons of the dorsal root ganglia (DRG), a collection of cell bodies of the afferent sensory fibres, which lie between adjacent vertebrae. The soma of the DRG neuron is spherical, and is located on a side branch of the main axon (receiving little synaptic input) and its isolation for techniques such as patch-clamp is relatively easy and can provide excellent recording conditions, making it an especially tractable model neuron. Additionally, changes in excitability of DRG neurons are of importance in a number of pathological conditions. Our understanding of the very different roles particular sodium channels play in influencing excitability of DRG neurons has progressed rapidly. In this review, we examine the roles of sodium channels in the excitability of DRG neurons.

Multiple sodium channel subtypes within DRG neurons

Conduction velocity of the dorsal root fibres has been associated with DRG cell size, classifying these neurons into four main groups: Aα, 30–55 m s⁻¹; Aβ, 14–30 m s⁻¹; Aδ, 2.2–8 m s⁻¹; and C, <1.4 m s⁻¹ (Harper & Lawson, 1985a,b). These fibres relay information from muscle and skeletal mechanoreceptors (Aα, Aβ), cutaneous and subcutaneous mechanoreceptors (Aβ, Aδ) and nociceptors (Aδ, C). The slower velocity fibres are generally associated with small cells, and the action potential duration is broader, often displaying an inflection or hump on the falling phase. Many of these characteristics have now been associated with expression of particular sodium channel subtypes in specific subclasses of DRG neurons. Studies of C-fibres (e.g. from sciatic nerve) have shown that they can generate a slow sodium-dependent spike that is resistant to tetrodotoxin (TTX), due to the presence of TTX-resistant (TTX-R) sodium channels (Gaumann et al. 1992; Kobayashi et al. 1993; Jeftinija, 1994; Buchanan et al. 1996) and, subsequently, to the specific isoform responsible for the current, Na_v1.8 (Akopian et al. 1996; Sangameswaran et al. 1996). Multiple studies have now shown that there are large variations in sodium current parameters in DRG neurons, linked to expression of a heterogeneous population of sodium channels (as summarized in Table 1; Kostyuk et al. 1981; Caffrey et al. 1992; Roy & Narahashi, 1992; Elliott & Elliott, 1993; Ogata & Tatebayashi, 1993; Rizzo et al. 1994; Rush et al. 1998; Cummins et al. 1999). Apart from pharmacological intervention using TTX, one can dissect the channels by using parallel methods of immunohistochemistry and electrophysiological recording, giving insight into the expression patterns and possible roles of the various channel isoforms. Using immunocytochemical techniques, large DRG cells have been shown to predominantly express TTX-sensitive (TTX-S) channels, such as Na_v1.1, Na_v1.6 and Na_v1.7, with some TTX-R Na_v1.8 expression (Black et al. 1996). Small cells, which are likely to be nociceptive in nature, express TTX-S channels (Black et al. 1996; Sangameswaran et al. 1997; Toledo-Aral et al. 1997), in conjunction with TTX-R Na_v1.8 and Na_v1.9 channels (Amaya et al. 2000; Fjell et al. 2000). These findings have been further corroborated using intracellular recording, together with immunohistochemistry, to show the distribution of channels in DRG neurons that give rise to particular fibre types (Fang et al. 2002, 2006; Djouhri et al. 2003a,b). Although it is not known whether the complement of sodium channels along, or at the terminals of, an axon are the same as at the cell body giving rise to that fibre, it is known that expression of multiple sodium channels, including TTX-R channels, is not limited to the cell body and extends along the fibres (Brock et al. 1998; Strassman & Raymond, 1999; Black et al. 2002a; Black & Waxman, 2002b; Rush et al. 2005a, 2006b), demonstrating the likely importance of these channels in conduction and fibre characteristics.

Table 1.

Summary of DRG sodium channels

Na+ channel Isoform	Type (Pharmacology/ kinetics)	Distribution	Unique biophysical characteristics in DRG	Role in AP generation
Nav1.1	TTX-S, fast	Widespread	Unknown	Unknown
Nav1.2	TTX-S, fast	Embryonic	Depolarized activation/inactivation for a TTX-S channel; can produce resurgent current	May maintain firing when misexpressed in MS damaged neurons
Nav1.3	TTX-S, fast	Embryonic	Rapid repriming, ramp current, persistent current	Ectopic firing when misexpressed in axotomy/SCI.
Nav1.4	TTX-S, fast	Not present	N/A	N/A
Nav1.5	TTX-R, fast	Embryonic	Not fully characterized	Unknown
Nav1.6	TTX-S, fast	Widespread	Rapid repriming; persistent current; can produce resurgent current	Maintains high frequency firing when present
Nav1.7	TTX-S, fast	Widespread	Slow onset of inactivation leading to ramp current, slow repriming	Ramp current amplifies small inputs
Nav1.8	TTX-R, slow	Widespread	Very depolarized activation/inactivation, rapid repriming	Major contributor to action potential upstroke and repetitive firing in small
Nav1.9	TTX-R,	Most small cells	Hyperpolarized activation, overlapping	May be involved in setting RMP,
	persistent	(esp. IB4+)	activation/inactivation curves, ultra-slow inactivation	amplification of inputs and/or maintaining activation of Nav1.8.

Trafficking and expression of sodium channels

Trafficking and expression of sodium channels can be regulated by association of channels with cofactors, in many cases in an isoform-specific manner. In DRG neurons, one such protein is annexin II/p11, which binds to the N-terminus of Na_v1.8 and facilitates insertion of functional Na_v1.8 channels into the cell membrane (Okuse et al. 2002). CAP-1A, a linker protein that binds clathrin and Na_v1.8, plays a complementary role, removing Na_v1.8 channels from the cell membrane (Liu et al. 2005). Contactin, a glycosyl-phosphatidylinositol (GPI)-anchored neuronal surface glycoprotein (Ranscht, 1988; Brummendorf et al. 1989; Gennarini et al. 1989), has been found to interact with Na_v1.2 via the β1-subunit and increases channel density at the plasma membrane in heterologous cells (Kazarinova-Noyes et al. 2001; Chen et al. 2004) and produces similar effects with Na_v1.3 and Na_v1.9 (Liu et al. 2001; Shah et al. 2004). In DRG neurons, contactin has been found to regulate current density of TTX-R sodium channels Na_v1.8 and Na_v1.9 in the subset of largely nociceptive, α-d-galactosyl lectin-binding IB4+ neurons, although TTX-S channels (Na_v1.6 and Na_v1.7) were unaffected (Rush et al. 2005a). TTX-S channels may instead be regulated by β2 subunits and this interaction has been shown to modulate the response to pain (Pertin et al. 2005; Lopez-Santiago et al. 2006). However, contactin may also play a role in the pathological re-emergence of Na_v1.3 in adult DRG neurons and accumulation of the channel in the neuroma of transected sciatic nerve (Shah et al. 2004). In addition to affecting plasma membrane channel density, the activation and inactivation kinetics of TTX-S currents are accelerated by β2, and other β-subunits are involved in modulation of Na_v1.8 (Shah et al. 2000; Vijayaragavan et al. 2004). Although some proteins may function primarily or solely as channel chaperones, such as annexin II/p11 with Na_v1.8 (Okuse et al. 2002), alteration of biophysical parameters can also occur with other cofactors. Fibroblast growth factor homologous factor (FHF) 2A and 2B have been demonstrated to be present in DRG neurons, and associate with Na_v1.6 to increase current density but also modulate the channel's biophysical properties, for instance, depolarizing steady-state inactivation (Wittmack et al. 2004; Rush et al. 2006b). Calmodulin can bind to the C-terminus of sodium channels (Herzog et al. 2003b) and can also modulate both the current density and biophysical properties of sodium currents generated by Na_v1.6 and Na_v1.8 in DRG neurons (Herzog et al. 2003b; Choi et al. 2006). Several of these proteins have been demonstrated to be coexpressed with their target interacting sodium channel, not only in the cell body but also along the nerve fibre (Wittmack et al. 2004; Liu et al. 2005; Rush et al. 2005a, 2006b). Thus, there are multiple sodium channel associated proteins in DRG neurons and associated fibres that control not only the density of different channel isoforms at the membrane but also some of the biophysical properties of the channels themselves.

Activation and inactivation characteristics

The slowly TTX-R inactivating sodium channel, now known to be Na_v1.8, which is specifically expressed within DRG and trigeminal neurons (Akopian et al. 1996; Sangameswaran et al. 1996), has more depolarized steady-state activation and inactivation (Fig. 1) characteristics than the TTX-S channels (Kostyuk, 1981; Caffrey et al. 1992; Roy & Narahashi, 1992; Elliott & Elliott, 1993; Ogata & Tatebayashi, 1993; Rizzo et al. 1994; Cummins & Waxman, 1997; Rush et al. 1998). DRG neurons are thus rather unique in having a mix of channels with an unusually wide range of varied characteristics. As we will see throughout this review, it has been possible over the last few years to examine the specific roles of different sodium channel isoforms in the firing behaviour of DRG neurons. Using knock-out mice, Na_v1.8 channels were shown to be essential for generation of single action potentials in most small DRG neurons and in fact generate about 80% of the inward current underlying the action potential upstroke in these cells (Renganathan et al. 2001). When Na_v1.8 was absent, the more hyperpolarized voltage-dependent properties of the TTX-S channels were revealed by inhibition of firing with even minor depolarization (Fig. 2). Although TTX-R channels are the major contributor to the action potential upstroke, TTX-S channels also play a significant part, especially around threshold, with a relatively minor involvement of high threshold calcium channels (Blair & Bean, 2002). The depolarized voltage dependence of inactivation of Na_v1.8, together with rapid recovery from inactivation (see below), also allows repetitive firing with sustained depolarization (Fig. 3) (Renganathan et al. 2001; Blair & Bean, 2003).

Figure 1. TTX-R currents carried by Na_v1.8 have more depolarized voltage dependence than TTX-S currents in DRG neurons.

Total sodium current in this DRG cell elicited using a holding potential of −100 mV and 25 ms step depolarizations to voltages ranging from −80 to +40 mV (A) can be pharmacologically separated on the basis of sensitivity to TTX (100 nm), allowing isolation of TTX-R currents (B) and by subtracting B from A, TTX-S currents (C). D, the voltage dependence of activation for TTX-R currents (▪) is depolarized compared to that of TTX-S currents (○) in DRG neurons. E, the voltage dependence of steady-state inactivation is more depolarized for the TTX-R currents (▪) than for TTX-S currents (○). The voltage dependence of inactivation was estimated by measuring currents elicited by 20 ms test pulses to −10 mV after 500 ms inactivating prepulses ranging from −130 to −10 mV. Adapted from data in Cummins & Waxman (1997) with permission; ©1997 by the Society for Neuroscience.

Figure 2. The TTX-R Na_v1.8 channel is a major contributor to the action potential upstroke in small DRG neurons.

A, TTX-R single action potentials recorded from small DRG neurons from a Na_v1.8+/+ mouse, in the presence of 250 nm TTX. B, a longer injection of current for 1 s generates a train of TTX-R action potentials in the same neuron. C, recordings under similar conditions as in (A), but in the absence of TTX, demonstrate that only graded potentials can be elicited from ∼80% of knockout Na_v1.8−/− mouse small DRG neurons. Full-sized action potentials cannot be recorded in these neurons, which lack Na_v1.8 expression. Around 20% of small Na_v1.8−/− DRG neurons have a more hyperpolarized RMP that allows overshooting action potentials (Da). Depolarizing the RMP in these neurons (Db, same cell as Da) inactivates the TTX-S channels and demonstrates the importance of Na_v1.8 in supporting action potential firing from a depolarized RMP. Adapted from Renganathan et al. (2001) with permission; © 2001 The American Physiology Society.

Figure 3. Na_v1.8 supports repetitive firing in small DRG neurons in response to sustained depolarization.

Long injections of current for 1 s (C) generate a train of action potentials in Na_v1.8+/+ small DRG neurons (A). The depolarized steady-state properties and rapid recovery from inactivation of the Na_v1.8 channel permit this firing despite a relatively depolarized RMP. In contrast, Na_v1.8−/− neurons are incapable of sustaining high frequency firing in response to identical stimuli (B). Adapted from Renganathan et al. (2001) with permission; © 2001 The American Physiology Society.

Na_v1.9, the second TTX-R sodium channel discovered within DRG neurons (Dib-Hajj et al. 1998), displays very slow activation and inactivation, with a large overlap between activation and steady-state inactivation that is centred near resting potential (Cummins et al. 1999). The kinetics of Na_v1.9 are so slow that it does not contribute to the action potential upstroke. In cells where it is present, Na_v1.9 enhances and prolongs the response to depolarizations that are subthreshold for action potential electrogenesis (Herzog et al. 2001) and lowers threshold for single action potentials and for repetitive firing (Baker et al. 2003). Because of its large predicted window current (a current produced by a significant crossover of activation and inactivation properties; Attwell et al. 1979) around RMP, it might be expected that Na_v1.9 might depolarize membrane potential (Herzog et al. 2001; Baker et al. 2003). Yet resting potential has been reported to be shifted in a depolarizing direction in Na_v1.9−/− knockout DRG neurons (Morisset et al. 2005; but see Priest et al. 2005), and in IB4– DRG neurons (which tend not to express Na_v1.9) compared to IB4+ neurons (which express high levels of Na_v1.9; Fang et al. 2006). A possible explanation of this apparent paradox is that Na_v1.9 might provide a return pathway for Na⁺ which is necessary for operation of Na⁺/K⁺-ATPase. Consistent with this speculation, block of persistent sodium conductance in optic nerve axons (Stys et al. 1993) and astrocytes (Sontheimer et al. 1994) produces a transient hyperpolarization as the depolarizing influence of the sodium conductance is blocked, followed by progressive depolarization due to failure of the Na⁺/K⁺-ATPase. Up-regulation of Na_v1.9 within DRG neurons with GTP is accompanied by a fall in threshold, repetitive firing in response to stimulation, and spontaneous activity in some cells even at relatively hyperpolarized levels, indicating that the effect of Na_v1.9 on threshold is not entirely due to the change in resting potential (Baker et al. 2003).

Repriming: recovery from inactivation

Several recent studies have shed light on the distinct patterns of recovery from inactivation (repriming) of the different sodium channel isoforms in DRG neurons. The TTX-S currents of intact large DRG neurons tend to have relatively fast repriming characteristics (Cummins & Waxman, 1997; Everill et al. 2001), which correlates with expression in these neurons of Na_v1.6 (Black et al. 1996), an isoform that displays this characteristic when expressed and studied in isolation in DRG (Herzog et al. 2003a; Rush et al. 2005b). In contrast, TTX-S currents in uninjured small DRG neurons, which include nociceptors, tend to have slow repriming (Elliott & Elliott, 1993; Cummins & Waxman, 1997; Rush et al. 1998; Black et al. 1999), similar to the behaviour of Na_v1.7 when expressed in cell lines or in a DRG background (Cummins et al. 1998; Herzog et al. 2003a). From this evidence, differential expression of Na_v1.6 or Na_v1.7 appears to endow these neurons with distinct TTX-S repriming properties. However, Na_v1.6 is expressed in all DRG cell sizes, including small cells giving rise to unmyelinated fibres (Black et al. 2002a; Rush et al. 2005a) and Na_v1.7 is also expressed in most DRG cells (Sangameswaran et al. 1997; Toledo-Aral et al. 1997). One possible explanation for this apparent discrepancy is that there are differences in the amount of non-functional channel protein for Na_v1.6 and Na_v1.7 channels in small and large DRG neurons. An alternative explanation is that functional properties of Na_v1.6 and/or Na_v1.7 are modified by other factors, such as associated proteins or kinases, that are differentially expressed in specific populations of DRG neurons. One likely candidate is fibroblast growth factor homologous factor 2A (FHF2A). This protein associates with Na_v1.6 via the C-terminus of the channel, is present in DRG neurons and strongly inhibits repriming of Na_v1.6, leading to a large accumulation of inactivation during repetitive stimulation (Rush et al. 2006b). Expression of FHF2A in small DRG neurons may help explain the anomalous repriming and channel expression observation outlined above. In addition, there may be as yet unknown cofactors that accelerate repriming of Na_v1.7 in larger DRG neurons. An emerging trend (see Cummins et al. 2001) is that the cellular background in which particular channels are expressed is an important determinant of the behaviour of sodium channel isoforms.

Recovery from inactivation is a critical channel characteristic that also plays a role in nerve injury. Chronic constriction injury (CCI) and axotomy of the sciatic nerve both trigger a down-regulation of Na_v1.8 and Na_v1.9 and an up-regulation of Na_v1.3 expression in nociceptive DRG neurons (Waxman et al. 1994; Dib-Hajj et al. 1996; Cummins & Waxman, 1997; Dib-Hajj et al. 1998; Cummins et al. 2000; Kim et al. 2002) that is accompanied by the emergence of a rapidly repriming TTX-S current in these cells (Fig. 4) (Cummins & Waxman, 1997). Na_v1.3 is normally only expressed during early stages of development and is practically undetectable in the adult rat nervous system (Beckh et al. 1989; Waxman et al. 1994; Felts et al. 1997). The change in channel expression is likely to be due to reduced access of axotomized DRG neurons to peripheral pools of neurotrophic factors such as nerve growth factor (NGF) and glial derived neurotrophic factor (GDNF), as the effects can be reversed by increasing their levels after nerve injury (Dib-Hajj et al. 1998; Fjell et al. 1999; Boucher et al. 2000; Cummins et al. 2000; Leffler et al. 2002).

Figure 4. A rapidly repriming TTX-S sodium current, largely attributable to Na_v1.3 channels, emerges in DRG neurons following transection of their peripheral axons.

A, family of TTX-S sodium current recordings from intact DRG neuron, showing recovery from inactivation at −80 mV. B, family of TTX-S sodium currents from a similar neuron 7 days after peripheral axotomy, demonstrating accelerated recovery from inactivation. C, single exponential fits showing accelerated repriming following peripheral axotomy. Modified from Black et al. (1999) with permission; © 2001 The American Physiology Society.

The emergence of Na_v1.3 in injured DRG neurons provides a clear example of the maladaptive effects that changes of sodium channel expression can have on a neuron. The TTX-S current of intact small, nociceptive DRG neurons normally recovers from inactivation slowly (Elliott & Elliott, 1993; Cummins & Waxman, 1997; Rush et al. 1998; Black et al. 1999). In contrast, the TTX-S current recorded from axotomized small DRG neurons reprimes rapidly (Cummins & Waxman, 1997), similar to the current produced when Na_v1.3 is expressed in a cell line or in DRG neurons (Cummins et al. 2001). Thus, high frequency firing may be boosted in axotomized DRG neurons by Na_v1.3. In addition, Na_v1.3 has slow onset of inactivation, allowing it to produce robust ramp current around resting membrane potential (RMP) (Cummins et al. 2001), and produces a persistent, as well as a transient current (Lampert et al. 2006). Therefore, previously subthreshold inputs may generate action potentials in the presence of Na_v1.3 (see Fig. 5). These changes are likely to contribute to the hyperexcitability and ectopic firing that have been observed (Wall & Gutnick, 1974; Lisney & Devor, 1987; Matzner & Devor, 1994) in spinal sensory neurons after injury.

Figure 5. Putative roles of different sodium channel subtypes in electrogenesis in small DRG neurons.

Representation of subthreshold responses and overshooting action potentials are shown from small intact and axotomized DRG neurons. Under normal conditions, Na_v1.7 (and Na_v1.9 in some cells) is likely to bring the neuron towards threshold, and Na_v1.8 is largely responsible for the overshooting action potential. Note the large depolarization necessary for action potential generation, due to the depolarized voltage dependence of activation of Na_v1.8. In contrast, axotomized DRG neurons are likely to have a more hyperpolarized threshold for overshooting action potential, in line with the predominance of TTX-S (Na_v1.1, Na_v1.3 and Na_v1.6) channels requiring less depolarization for activation and the boosted ramp currents with the presence of Na_v1.3. Note that Na_v1.8 and Na_v1.9 are no longer involved due to down-regulation of expression after axotomy.

Slow inactivation

In addition to fast inactivation, DRG sodium channels can also undergo slow inactivation (Ogata & Tatebayashi, 1992; Rush et al. 1998), a process that occurs over a longer time course. The mechanism is thought to be separate from the III–IV linker associated with fast inactivation, as the phenomenon is maintained in channels of squid axon, despite removal of fast inactivation with pronase (Rudy, 1978). The exact structural rearrangements are still being elucidated, and appear to involve many different regions of the channel (for reviews see Goldin, 2003; Ulbricht, 2005). Interestingly, different sodium channel isoforms undergo different levels of slow inactivation (Vilin et al. 2001) and thus the mixed populations of channels in DRG neurons are likely to be affected differently by RMP and periods of depolarization. In comparisons of small DRG TTX-S and TTX-R currents, despite the more depolarized fast inactivation characteristics, TTX-R channels had quicker and much more complete slow inactivation at depolarized voltages (Rush et al. 1998; Blair & Bean, 2003) and this perhaps explains the differential block by agents sometimes used in the treatment of neuropathic pain, such as phenytoin and carbamazapine (Rush & Elliott, 1997; Cardenas et al. 2006). The midpoint of TTX-R slow inactivation is actually more hyperpolarized than that for fast inactivation (Blair & Bean, 2003), in marked contrast to the reverse situation commonly found for TTX-S currents (Kuo & Bean, 1994; Fleidervish et al. 1996; Fazan et al. 2001). Build-up of slow inactivation is likely to be responsible for the use-dependent inhibition of TTX-R currents on repetitive stimulation (Rush et al. 1998; Blair & Bean, 2003; Tripathi et al. 2006) and the cell-to-cell variations in TTX-R current properties seen in these studies might reflect regulation of Na_v1.8 by calmodulin (Choi et al. 2006). Interestingly, use-dependent slow inactivation of Na_v1.8 current is stronger and develops more rapidly in IB4+ DRG neurons, compared to IB4– DRG neurons, and recovery from slow inactivation is slower in IB4+ neurons (Choi et al. 2007), adding to evidence (Stucky & Lewin, 1999; Braz et al. 2005; Fang et al. 2006) that these subclasses of DRG neurons are functionally distinct.

Resurgent current

Current that occurs upon repolarization is termed resurgent current and was first recorded from cerebellar Purkinje neurons (Raman & Bean, 1997a). It is a transient current that displays slow re-activation and inactivation upon rapid repolarization. Initially, resurgent current was linked with the presence of Na_v1.6 in Purkinje neurons (Raman et al. 1997b). Evidence to date suggests that at least some subpopulations of DRG neurons can produce resurgent current (Cummins et al. 2005; Rush et al. 2005b). About 60% of DRG neurons transfected with Na_v1.6 produce resurgent currents attributable to this channel (Cummins et al. 2005). Other studies have led to the suggestion that other sodium channel isoforms may also be able to produce a resurgent current as it can be recorded from neurons that do not express Na_v1.6 (Afshari et al. 2004; Do & Bean, 2004). In line with this is the observation that approximately 8% of DRG neurons transfected with Na_v1.2 can produce resurgent current attributable to this channel isoform (Rush et al. 2005b).

With normal channel isoform expression in the adult, the resurgent current may be limited to large DRG neurons (Cummins et al. 2005). The cofactor(s) that permit Na_v1.6 and Na_v1.2 to produce resurgent current in some cell types, but not in others, are still under study. Phosphorylation of the sodium channels may play a role (Grieco et al. 2002) and open channel block by the sodium channel β4 subunit appears to be involved, for the Na_v1.6 channel at least (Grieco et al. 2005). The specific role of the resurgent current in DRG neurons has still to be elucidated but in other neurons it has been associated with rapid burst firing in response to large depolarizations (Raman & Bean, 1997a; Khaliq et al. 2003; Swensen & Bean, 2003; Magistretti et al. 2006).

Ramp currents

As we have discussed, Na_v1.8 carries a current that has very depolarized voltage dependence of activation and inactivation and is extremely resistant to depolarization. However, TTX-S currents do still play a role in the action potential, especially around threshold (Blair & Bean, 2002). Na_v1.7 is one of the major candidates that has been suggested to amplify subthreshold generator potentials by producing a prominent ramp current (response to small, slow depolarizing stimuli), largely due to its slow onset of closed-state inactivation (Cummins et al. 1998). It is thought that under conditions where there is slow depolarization of a neuron, this particular channel would tend to remain available for activation, in comparison with other channels that are able to activate only at relatively hyperpolarized potentials. This property would be predicted to make Na_v1.7 a significant source of ramp currents and this isoform has indeed been shown to produce a robust ramp current in response to slow depolarizations (Cummins et al. 1998; Herzog et al. 2003a) (see Fig. 6). More recently, mutations in Na_v1.7, associated with the neuropathic pain syndrome erythromelalgia (Cummins et al. 2004; Yang et al. 2004; Dib-Hajj et al. 2005; Drenth et al. 2005; Han et al. 2005), have helped to demonstrate its role as a threshold channel that can interact with Na_v1.8 to produce hyperexcitability and repetitive firing behaviour of nociceptive neurons (Dib-Hajj et al. 2005; Rush et al. 2006a). Na_v1.7 appears to bring the neuron closer to the potential needed for activation of Na_v1.8, thereby increasing excitability. In marked contrast, when the effects of an erythromelalgia mutation were examined in a cell type that lacks Na_v1.8 (sympathetic ganglion neurons), a reduction in excitability was seen (Rush et al. 2006a), presumably due to inactivation of the TTX-S sodium channels essential for the action potential upstroke in those neurons. Subsequent addition of Na_v1.8 into sympathetic neurons allowed these cells to fire action potentials despite depolarization of resting membrane potential induced by the mutant Na_v1.7 channels, demonstrating how sodium channel isoforms with different biophysical characteristics can interact with one another (Fig. 7). These data may help to explain both the pain caused by mild stimulation (due to hyperexcitability of nociceptive DRG neurons) and also the sympathetic dysfunction associated with this disorder (flushing of the extremities due to loss of vasoconstrictive tone) which is due to hypoexcitability of sympathetic ganglion neurons (Rush et al. 2006a).

Figure 6. Distinct biophysical characteristics of the Na_v1.7 sodium channel shape its role in DRG neurons.

Trains of 50 Hz stimulation show that sodium channel Na_v1.7 is unable to follow and sustain high frequency firing (B), when compared to, for example, Na_v1.4 (A). Thus Na_v1.7 is unlikely to play a major role in the action potential upstroke during repetitive firing. Using ramp stimulation to mimic small graded subthreshold potentials, Na_v1.4 produces only a small response (C), in marked contrast to the large ramp current evoked with Na_v1.7 (D), similar to those that can be recorded from small DRG neurons (E) (the asterisk marks a second component, blocked by cadmium and therefore likely to be due to calcium influx). F, Na_v1.7 has much slower onset of inactivation than Na_v1.4. Because of this, small changes in membrane potential, such as generator potentials, produce less closed-state inactivation in Na_v1.7. Resistance to this form of inactivation enables Na_v1.7 to respond to small, slow depolarizations close to RMP, boosting these depolarizations and bringing the cell closer to action potential threshold. Adapted from Cummins et al. (1998) with permission; ©1998 by the Society for Neuroscience.

Figure 7. The erythromelalgia Na_v1.7 mutation L858H inhibits firing in SCG neurons, which can be rescued by coexpression of Na_v1.8 channels.

When Na_v1.8 is coexpressed with the Na_v1.7 mutant L858H, current threshold and action potential overshoot are restored, even though the depolarization of RMP induced by L858H persists. A, suprathreshold action potentials recorded from representative superior cervical ganglion (SCG) neurons transfected with WT (blue), L858H (red) and L858H plus Na_v1.8 (green) channels. B, depolarized RMP in cells with L858H channels is maintained with coexpression of Na_v1.8 (P > 0.05). Dashed line indicates RMP in cells expressing WT Na_v1.7 alone. C, current threshold for action potential firing is reduced by L858H coexpression with Na_v1.8, when compared to L858H alone (P < 0.05). D, action potential overshoot in SCG neurons with L858H channel is increased when Na_v1.8 is coexpressed with L858H (41.5 ± 4.6 mV, n = 17; P < 0.05). Thus, the introduction of Na_v1.8, with depolarized activation and inactivation characteristics, allows SCG neurons to fire full overshooting action potentials, despite the depolarization induced by the L858H erythromelalgia mutation in Na_v1.7. Adapted from Rush et al. (2006a) with permission; © 2006 National Academy of Sciences, USA.

Na_v1.3 also produces a prominent ramp current and this also amplifies small depolarizing stimuli. This factor may contribute to hyperexcitability of axotomized DRG neurons in which expression of this channel isoform is up-regulated (Cummins & Waxman, 1997).

Sodium channel combinations and interactions

Thus far, we have discussed many of the specific biophysical characteristics that define particular sodium channel isoforms, how they behave in the DRG neuron background and what their individual influence may be on the neurons and neuronal processes where they are expressed. However, in some conditions, there are not only long-term chronic changes in expression of multiple sodium channels, but also shorter term acute alterations in behaviour of existing channels, in response to changes in their environment. Thus, in order to understand the overall likely effects on firing behaviour, it is important to consider how the rise in expression of one channel or change in biophysical characteristics of another might relate to each other. For example, in models of inflammatory pain such as carrageenan injection into the hindpaw there is an up-regulation of TTX-S channels Na_v1.3 and Na_v1.7 in DRG after a few days, together with a parallel increase in the TTX-S current (Black et al. 2004). This is in addition to an increased expression of Na_v1.8 and a boosted TTX-R current (Tanaka et al. 1998; Black et al. 2004). In contrast, expression of Na_v1.6 and Na_v1.9 are relatively unaffected in the inflammatory response. These changes might amplify previously subthreshold inputs (due to up-regulation of Na_v1.3 and Na_v1.7) and help in sustaining repetitive firing under prolonged depolarization (Na_v1.3 and Na_v1.8).

Na_v1.8 is also likely to be involved in acute inflammatory pain as administration of agents such as prostaglandin E2 (PGE2) and 5-HT produces an increase in current amplitude and a hyperpolarizing shift in activation, both likely to increase excitability (England et al. 1996; Gold et al. 1996; Cardenas et al. 1997, 2001). Recent evidence also points towards the involvement of Na_v1.9 in the inflammatory response. Acute administration of inflammatory mediator PGE2 causes a large increase in the amplitude of Na_v1.9 (Rush & Waxman, 2004), a channel which, when knocked out in the mouse, produces deficits in the inflammatory response but not in neuropathic pain models (Priest et al. 2005). These data suggest that Na_v1.9 may play a significant role in the increased excitability of nociceptive fibres during inflammation, either by effects on resting potential, by sustaining depolarization, or by lowering threshold (Herzog et al. 2001; Baker et al. 2003). However, in another inflammatory model, induced jejunitis was linked to changes in Na_v1.8 but knocking out Na_v1.9 had no effect (Hillsley et al. 2006). There is still much to be done to fully understand the intricate roles of sodium channel isoforms in the variety of pain models and to translate these results to the clinic.

Overview: does complexity of sodium channel expression represent a therapeutic opportunity?

Although the expression of an especially large repertoire of sodium channel isoforms makes DRG neurons complex, the accessibility and spherical shape of the these cells, and their involvement in a number of readily modelled disease states involving sodium channels, have made them a very tractable model for the study of the roles of sodium channels in electrogenesis. Excitability of DRG neurons can be affected by changes during development and a variety of pathologies and injuries. Sodium channels, which were once thought to be uniform in terms of biophysical properties and simply responsible for the rising phase of an action potential, have now been shown to be able to play multiple roles in electrogenesis, and thus can finely tune the behaviour of excitable cells. The presence of multiple sodium channel isoforms within DRG neurons suggests the possibility of pharmacologically targeting specific channel subtypes, thus possibly achieving therapeutic activity whilst limiting undesirable side-effects. Changes in expression levels or physiological properties of specific sodium channel isoforms are likely to contribute to altered function of DRG neurons in a variety of pathologies and these isoforms may therefore be tractable targets for subtype specific modulators.