Nav1.3 Sodium Channels: Rapid Repriming and Slow Closed-State Inactivation Display Quantitative Differences after Expression in a Mammalian Cell Line and in Spinal Sensory Neurons

Abstract

Although rat brain Nav1.3 voltage-gated sodium channels have been expressed and studied in Xenopus oocytes, these channels have not been studied after their expression in mammalian cells. We characterized the properties of the rat brain Nav1.3 sodium channels expressed in human embryonic kidney (HEK) 293 cells. Nav1.3 channels generated fast-activating and fastinactivating currents. Recovery from inactivation was relatively rapid at negative potentials (ϽϪ80 mV) but was slow at more positive potentials. Development of closed-state inactivation was slow, and, as predicted on this basis, Nav1.3 channels generated large ramp currents in response to slow depolarizations. Coexpression of ␤3 subunits had small but significant effects on the kinetic and voltage-dependent properties of Nav1.3 currents in HEK 293 cells, but coexpression of ␤1 and ␤2 subunits had little or no effect on Nav1.3 properties. Nav1.3 channels, mutated to be tetrodotoxin-resistant (TTX-R), were expressed in SNS-null dorsal root ganglion (DRG) neurons via biolistics and were compared with the same construct expressed in HEK 293 cells. The voltage dependence of steadystate inactivation was ϳ7 mV more depolarized in SNS-null DRG neurons, demonstrating the importance of background cell type in determining physiological properties. Moreover, consistent with the idea that cellular factors can modulate the properties of Nav1.3, the repriming kinetics were twofold faster in the neurons than in the HEK 293 cells. The rapid repriming of Nav1.3 suggests that it contributes to the acceleration of repriming of TTX-sensitive (TTX-S) sodium currents that are seen after peripheral axotomy of DRG neurons. The relatively rapid recovery from inactivation and the slow closed-state inactivation kinetics of Nav1.3 channels suggest that neurons expressing Nav1.3 may exhibit a reduced threshold and/or a relatively high frequency of firing.

Altered Sodium Channel Expression in Second-Order Spinal Sensory Neurons Contributes to Pain after Peripheral Nerve Injury

“…Upregulation of Na v 1.3 is functionally important because, Na v 1.3 recovers (reprimes) rapidly from inactivation, a property that poises neurons to produce highfrequency activity (Cummins and Waxman, 1997;Black et al, 1999;Cummins et al, 2001). Increased expression of Na v 1.3 occurs in DRG neurons after injury to the sciatic nerve (Waxman et al, 1994;DibHajj et al, 1996;Black et al, 1999;Kim et al, 2001) and in facial motor neurons after transection of the facial nerve (Iwahashi et al, 1994), but expression of Na v 1.3 is not increased in spirally axotomized cortical pyramidal neurons (Hains et al, 2002).…”

“…18S ribosomal RNA (rRNA) was used as an endogenous control to normalize expression levels. Na v 1.3 cDNA was obtained from human embryonic kidney 293 cells stably transfected with an Na v 1.3 construct (Cummins et al, 2001). Standards and unknowns were amplified in quadruplets.…”

“…However, expression of Na v 1.3 is upregulated within DRG neurons after their axotomy by sciatic nerve transection (Waxman et al, 1994;Black et al, 1999) and after chronic constriction injury (CCI) of the sciatic nerve in adult rats (DibHajj et al, 1999). This is functionally important, because Na v 1.3 produces a rapidly repriming TTX-S current that permits neuronal firing at higher-than-normal frequencies within DRG neurons (Cummins and Waxman, 1997;Cummins et al, 2001). To date, however, the question of whether peripheral nerve injury triggers changes in sodium channel expression within higherorder sensory neurons has not been examined.…”

Familial Hemiplegic Migraine

“…While haploinsufficiency for SCNA1A and consequent reduced excitability of inhibitory neurons might explain seizure susceptibility in most cases of SMEI, the mechanism underlying a milder epileptic disorder (generalized epilepsy with febrile seizures plus: GEFSϩ), that is caused by missense mutations in SCNA1A leading (in most cases) to gain-of-function alterations in channel activity remains unclear 87 (see also Heron et al 91 ). Considering the high sensitivity of the functional properties of Na V channels to the cell background (e.g., compare Cummins et al 92 ) and the broad expression of Na V 1.1 channels in excitatory and inhibitory neurons, one may hypothesize that different types of SCNA1A mutations may affect differently different subpopulations of neurons, as shown in the case of CACNA1A mutations where loss-of-function of Ca V 2.1 mainly affects cerebellar function, whereas gain-of-function of Ca V 2.1 mainly affects cortical function.…”

Jingzhaotoxin-III, a Novel Spider Toxin Inhibiting Activation of Voltage-gated Sodium Channel in Rat Cardiac Myocytes

“…However, it is the divergent residues among the sequences of these VGSC isoforms that determine their response to distinct ligands. For instance, after tyrosine 371 is substituted by serine in rNav1.6 and rNav1.3 (wild types), which are TTX-S phenotypes, the mutants become resistant to TTX (7,8).…”