Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Nav1.7 sodium channels in primary afferents.

Induction of peripheral inflammation increases the expression of the Nav1.7 sodium channel in sensory neurons, potentially increasing their excitability. Peripheral inflammation also produces hyperalgesia in humans and an increase in nociceptive responsiveness in animals. To test the relationship between these two phenomena we applied a recombinant herpes simplex-based vector to the hindpaw skin of mice, which encoded both green fluorescent protein (GFP) as well as an antisense sequence to the Nav1.7 gene. The hindpaw was subsequently injected with complete Freund's adjuvant to induce robust inflammation. Application of the vector, but not a control vector encoding only GFP, prevented an increase in Nav1.7 expression in GFP-positive neurons and prevented development of hyperalgesia in both C and Adelta thermonociceptive tests. These results provide clear evidence of the involvement of an increased expression of the Nav1.7 channel in nociceptive neurons in the development of inflammatory hyperalgesia.

The effect of the mouse mutation claw paw on myelination and nodal frequency in sciatic nerves.

Despite the biophysical and clinical importance of differentiating nodal and internodal axolemma, very little is known about the process. We chose to study myelination and node of Ranvier formation in the hypomyelinating mouse mutant claw paw (clp). The phenotype of clp is delayed myelination in the peripheral nervous system. The specific defect is unknown but is thought to arise from a breakdown in the complex signaling mechanism between axon and Schwann cell. Myelination was assessed in sciatic nerve cross sections from adult and postnatal day 14 (P14) heterozygous and homozygous clp mice. Antibodies to P0, myelin-associated glycoprotein (MAG), and neural cell adhesion molecule were used to assess the stage of myelination. P14 homozygous clp mice showed an atypical staining pattern of immature myelin, which resolved into a relatively normal pattern by adulthood. Sodium channel clustering and node of Ranvier frequency were studied in whole-mount sciatic nerves with sodium channel and MAG antibodies. P14 homozygous clp nerves again showed an atypical, immature pattern with diffuse sodium channel clusters suggesting nodal formation was delayed. In the adult, homozygous clp sciatic nerves displayed dramatically shortened internodal distances. The data from this study support the hypotheses that node of Ranvier formation begins with the onset of myelination and that the number and location of nodes of Ranvier in the sciatic nerve are determined by myelinating Schwann cells.

Contribution of sialic acid to the voltage dependence of sodium channel gating. A possible electrostatic mechanism.

A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium channels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line (lec2). The steady-state half-activation voltages (Va) of channels under each condition of reduced sialylation were approximately 10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for channels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements.

Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons.

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.

Clustering of Na+ channels and node of Ranvier formation in remyelinating axons.

Polyclonal antibodies were raised against a well conserved region of the vertebrate Na+ channel and were affinity purified for use in immunocytochemistry. Focal demyelination of rat sciatic axons was initiated by an intraneural injection of lysolecithin and Na+ channel clustering was followed at several stages of myelin removal and repair. At 1 week post-injection axons contained long, fully demyelinated regions. Na+ channel clusters appeared only at heminodes forming the borders of these zones, and at widely spaced isolated sites that may represent former nodes of Ranvier. Over the next few days proliferating Schwann cells adhered to axons and began to extend processes. Clusters of Na+ channels appeared at the edges of these structures. As the Schwann cells elongated, the clusters seemed to move with them, since they remained at edges and the distance between aggregates increased. Clusters associated with different Schwann cells ultimately approached each other and appeared to fuse. Na+ channels then coalesced further at these sites, forming new nodes of Ranvier in regions that previously were internodal. If Schwann cell proliferation were blocked by mitomycin, no new clusters of Na+ channels appeared within internodes. Under these conditions, heminodal clusters remained visible at 1 week postinjection, but by 2 weeks they were no longer detectable, suggesting that proliferating Schwann cells are required for their maintenance. Clusters at normal nodes of Ranvier remained. It is concluded that Na+ channel aggregation and mobility in demyelinated nerve fibers is controlled by adhering Schwann cells, resulting in the formation of stable new nodes of Ranvier during remyelination.