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Sodium channels Nav1.2 and Nav1.6 are both normally expressed along premyelinated and myelinated axons at different stages of maturation and are also expressed in a subset of demyelinated axons, where coexpression of Nav1.6 together with the Na+/Ca2+ exchanger is associated with axonal injury. It has been difficult to distinguish the currents produced by Nav1.2 and Nav1.6 in native neurones, and previous studies have not compared these channels within neuronal expression systems. In this study, we have characterized and directly compared Nav1.2 and Nav1.6 in a mammalian neuronal cell background and demonstrate differences in their properties that may affect neuronal behaviour. The Nav1.2 channel displays more depolarized activation and availability properties that may permit conduction of action potentials, even with depolarization. However, Nav1.2 channels show a greater accumulation of inactivation at higher frequencies of stimulation (20–100 Hz) than Nav1.6 and thus are likely to generate lower frequencies of firing. Nav1.6 channels produce a larger persistent current that may play a role in triggering reverse Na+/Ca2+ exchange, which can injure demyelinated axons where Nav1.6 and the Na+/Ca2+ exchanger are colocalized, while selective expression of Nav1.2 may support action potential electrogenesis, at least at lower frequencies, while producing a smaller persistent current.
Voltage-gated sodium channels are critical for electrogenesis in excitable cells; at least nine distinct sodium channel isoforms have been identified in mammals (Goldin et al. 2000). It is possible to study the physiological properties of these channels in isolation in cell lines or in oocytes, but the appropriate ensemble of associated proteins, such as β-subunits (Isom et al. 1994) may not be present, and thus it is not clear whether the characteristics recorded in these expression systems accurately reflect the in vivo properties of the channels.
In this study, we have characterized and compared sodium channels Nav1.2 and Nav1.6 in a mammalian, neuronal cell background using the technique (Cummins et al. 2001; Herzog et al. 2003) of expressing TTX-resistant (TTX-R) versions of these channels in dorsal root ganglia (DRG) neurones from Nav1.8-null mice (Akopian et al. 1999), which permits recording in isolation from other sodium currents. Our rationale for comparing these two channels arises from their sequential expression during development of myelinated axons and their altered patterns of expression in demyelinated axons under pathological conditions. Nav1.2 is present in premyelinated CNS neurones and at immature nodes of Ranvier, before a transition to expression of Nav1.6 at mature nodes (Boiko et al. 2001; Kaplan et al. 2001), but to date, the question of whether expression of Nav1.6, rather than Nav1.2, might be functionally advantageous has not been explored.
The comparative physiology of Nav1.2 and Nav1.6 may also be relevant to demyelinated axons. Both Nav1.2 and Nav1.6 are expressed along demyelinated axons in white matter from mice with experimental autoimmune encephalomyelitis (EAE) (Craner et al. 2003, 2004a) and in human white matter from acute multiple sclerosis (MS) plaques (Craner et al. 2004b). Nav1.6, which has been shown to produce persistent current (Smith et al. 1998; Burbidge et al. 2002), is colocalized with the Na+/Ca2+ exchanger in injured axons, while Nav1.2 is expressed, often together with the Na+/Ca2+ exchanger, along demyelinated axons that do not show signs of injury in EAE (Craner et al. 2004a) and in MS (Craner et al. 2004b), consistent with the suggestion that a persistent sodium conductance can drive reverse Na+/Ca2+ exchange that contributes to axonal degeneration (Stys et al. 1992; Stys et al. 1993). Because dysmyelinated axons that express Nav1.2 are much less susceptible to this type of injury (Waxman et al. 1990), we hypothesized that Nav1.2 would produce a smaller persistent current than Nav1.6.
In our comparison of Nav1.2 and Nav1.6, we have also examined resurgent current, which was initially recorded from Purkinje neurones and linked to the presence of Nav1.6 (Raman & Bean, 1997). The resurgent current is a transient current that displays slow activation and inactivation upon rapid repolarization. Because more recent studies have led to the suggestion that other (but unspecified) sodium channel isoforms may also be able to produce resurgent current (Afshari et al. 2004; Do & Bean, 2004; Grieco & Raman, 2004), we compared the ability of Nav1.2 and Nav1.6 to produce resurgent current.
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This study examined the voltage-dependent and kinetic properties of two of the TTX-S sodium channels, Nav1.2 and Nav1.6, which are expressed along premyelinated, myelinated and demyelinated axons. Specific blockers for these channels are not available and thus it has been impossible to characterize their endogenous currents in isolation. We have been able to study the currents produced by these different channel isoforms and directly compare their properties in a mammalian, neuronal cell background. This comparison, in conditions that mimic the in vivo situation, provides insight into how the properties of particular channels may influence the behaviour of neurones where these channels are expressed.
In this study, the voltage-dependent properties of activation and availability of the transient sodium currents were around 15 mV more depolarized for Nav1.2R than Nav1.6R. This is consistent with results from previous studies in which Nav1.2 and Nav1.6 were expressed in mammalian cell lines though under non-identical conditions, which taken together suggest that Nav1.6 activates and inactivates at more hyperpolarized potentials than Nav1.2 (Xie et al. 2001; Burbidge et al. 2002). As with a previous study from our group of sodium channel physiology, which compared Nav1.6 and Nav1.7 (Herzog et al. 2003), the parallel shift of activation and availability curves for Nav1.2 versus Nav1.6 currents is consistent with the hypothesis (Chahine et al. 1994) that activation and inactivation are linked. More robust expression of Nav1.2, which occurs in some demyelinated axons (Craner et al. 2003, 2004a,b), could endow a neuronal membrane with the capability to fire action potentials from resting membrane potentials that are much more depolarized than normal. With tissue damage, the external potassium concentration may rise; this could depolarize neurones and the expression of Nav1.2 could help maintain firing in such a situation, although the change in resting membrane potential (if any) in demyelinated axons compared to normal axons is not known. However, if both isoforms were present, the combination of channels could provide a cross-over current and make the axon unstable, which might explain why Nav1.2 has to be completely replaced by Nav1.6 at nodes of Ranvier along normal myelinated axons (Boiko et al. 2001; Kaplan et al. 2001).
It has been suggested that Nav1.6 produces a large persistent current (Smith et al. 1998; Burbidge et al. 2002), but no direct comparison with other sodium channel isoforms has been carried out. In our experiments, both Nav1.2R and Nav1.6R produced a significant persistent current but the Nav1.6R current was two-fold larger. Consistent with production of a robust persistent current by Nav1.6, a large, TTX-S persistent sodium current is present within DRG neurones (Baker & Bostock, 1997), which express Nav1.6 at high levels (Black et al. 1996). Nav1.6 is coexpressed with the Na+/Ca2+ exchanger (NCX) in injured axons while Nav1.2 and the NCX tended to be expressed along axons that did not show signs of injury in mice with EAE (Craner et al. 2004a) and in human MS tissue (Craner et al. 2004b). Persistently activated sodium channels have been proposed to drive an injury cascade via reverse Na+/Ca2+ exchange (Stys et al. 1992, 1993). Conversely, dysmyelinated axons, which express Nav1.2 (Westenbroek et al. 1989; Boiko et al. 2001), have been shown to be much less susceptible to injury triggered by sodium channels (Waxman et al. 1990). The results of the present study support the notion that expression of Nav1.6, which occurs in some demyelinated axons in EAE (Craner et al. 2003, 2004a) and in MS (Craner et al. 2004b), could lead to a larger persistent current than with the other sodium channel isoform expressed along demyelinated axons (Nav1.2), and could initiate a damaging flux of ions, contributing to axonal degeneration.
Endogenously expressed TTX-S and TTX-R sodium channels in DRG neurones produce currents with very different repriming or recovery from inactivation (Elliott & Elliott, 1993; Rush et al. 1998). Different TTX-S currents can also show distinct repriming characteristics following a single depolarizing stimulus, where those in small DRG neurones have slow recovery from inactivation and those in large neurones, fast (Cummins & Waxman, 1997; Everill et al. 2001). This may be due to differential expression of Nav1.7 and Nav1.6 in these neurones (Herzog et al. 2003), endowing these neurones with slow and fast repriming, respectively. Consistent with previous studies from both a cell line and neurones (Burbidge et al. 2002; Herzog et al. 2003), we confirm that repriming of Nav1.6R after a single depolarizing stimulus is fast. Previous work on Nav1.2 in an HEK293 cell line reported recovery from inactivation that was around three times slower than that for Nav1.6 (O'Leary, 1998). In contrast to this, in our direct comparison within a mammalian neuronal background, we found that repriming during the first 50 ms following a single depolarizing stimulus was more rapid for the Nav1.2R isoform. Although this result may seem surprising given that Nav1.6 has been linked with rapid, burst firing (Raman et al. 1997; Swensen & Bean, 2003), we found that Nav1.6 was able to more faithfully follow high-frequency repetitive stimuli, a characteristic described below.
Slow development of inactivation, which characterizes Nav1.7 (Cummins et al. 1998; Herzog et al. 2003) and Nav1.3 (Cummins et al. 2001) channels, is associated with more robust responses to slow depolarizing inputs (Cummins et al. 1998). In contrast, Nav1.6 displays relatively rapid onset of inactivation (Herzog et al. 2003), and this was confirmed in the present study. Nav1.2R showed slightly slower onset (time constants of ∼30 ms instead of ∼10 ms), but this was still very much quicker than for Nav1.3 and Nav1.7 channels (∼150 ms) (Cummins et al. 1998; Herzog et al. 2003). It could therefore be predicted that Nav1.2 would respond in a similar way to Nav1.6 and activate in response to large depolarizations rather than small, slow ones.
Resurgent current was first described in Purkinje neurones and was attributed to the presence of Nav1.6 in these cells because the current was minimal (∼10% of normal) in Nav1.6-null (med) mice (Raman & Bean, 1997). The current can be inhibited by phosphorylation blockers (Grieco et al. 2002) and has been associated with rapid burst firing in response to large depolarizations (Raman et al. 1997; Khaliq et al. 2003; Swensen & Bean, 2003). More recent studies have led to recognition that other sodium channel isoforms may be able to produce resurgent current as well. The suggestion that this current is produced by an endogenous open-channel blocker is supported by the demonstration of re-emergence of robust resurgent current in Nav1.6-null Purkinje neurones, when inactivation of the transient current was slowed (Grieco & Raman, 2004). Further evidence was provided by recordings of the current in subthalamic nuclei (Do & Bean, 2004) and in granule cells, unipolar brush cells and cerebellar nuclei (Afshari et al. 2004), although in the latter study, specific recording conditions were manipulated to observe the currents. Grieco et al. (2005) provided evidence which supports a role of the cytosolic tail of the sodium channel β4 subunit (Yu et al. 2003), as the open channel blocker. The evidence for a resurgent current in spinal neurones is less well documented, with studies showing either no occurrence (Pan & Beam, 1999) or its presence only in a subpopulation of large DRG neurones (Cummins et al. 2003). Our present work demonstrates that Nav1.6R can produce resurgent current in around 20% of transfected small DRG neurones, and that this current activates over a potential range similar to that found previously in Purkinje neurones (Raman & Bean, 1997). When either Nav1.4 or Nav1.7 were expressed in DRG neurones, no resurgent current could be detected in any cells (Cummins et al. 2003). In contrast, we show here that Nav1.2R can produce resurgent current in a small number of neurones. The data reported in this study may underestimate the occurrence of Nav1.2R resurgent current because of the smaller size of the transient currents in these cells, since this made detection of the resurgent current much more difficult. Our study confirms that another isoform, in addition to Nav1.6, is capable of producing a resurgent current within some cells in at least one neuronal background. We suggest that Nav1.2 could be the sodium channel subtype that gives rise to resurgent current in neurones that do not express Nav1.6 (Afshari et al. 2004; Do & Bean, 2004).
The recovery from inactivation experiments in our study showed that Nav1.2R and Nav1.6R had fast time constants for recovery from a single stimulus and, if anything, Nav1.2R reprimed faster under those test conditions, using a holding potential of −100 mV. However, Nav1.6 has been associated with rapid, burst firing (Raman et al. 1997; Swensen & Bean, 2003) and we therefore examined the response of the currents to trains of stimulation, from a holding potential of −80 mV, to more closely mimic a possible in vivo situation. Our data demonstrate that both channels can follow repetitive stimulation up to around 10 Hz. However, at higher frequencies of 20 and 100 Hz, Nav1.6 currents were able to maintain a more robust transient current, from this holding potential, which may influence other biophysical parameters of the channels, such as slow inactivation. This implies that Nav1.6 may be able to follow high-frequency trains more faithfully than Nav1.2. The greater amount of persistent current produced by the Nav1.6 channel could be, in part, responsible for the ability to follow high frequencies. A recent paper also compared these channels using trains of stimulation, using an oocyte expression system (Zhou & Goldin, 2004). The authors describe a use-dependent potentiation of the Nav1.6 channel with high-frequency trains, although a potentiation of endogenous sodium currents in mammalian cells has not been reported in the literature, and we found no evidence for potentiation in the current study.
In this paper, we have studied two sodium channel subtypes expressed in mammalian, neuronal cells and have shown that there are several important differences in their properties. The Nav1.2 channel may provide a basis for the firing of action potentials, even with strong depolarization, although the kinetics of Nav1.2 may be best suited to low frequencies of opening. Because Nav1.2 produces only a small persistent current, selective expression of this channel in some demyelinated axons may provide a basis for maintaining firing capability, at least at lower frequencies, while limiting the sustained Na+ influx that has been shown (Stys et al. 1992) to drive damaging reverse Na+/Ca2+ exchange. In contrast, Nav1.6 expression may allow neurones to fire at high frequencies. However, the larger persistent current produced by Nav1.6 may play a role in a damaging injury cascade, when coexpressed with the Na+/Ca2+ exchanger in demyelinated axons (Craner et al. 2004a,b).