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Introduction

Voltage-gated sodium channels (VGSCs) are responsible for the rising phase of the action potential and play a key role in mediating electrical activity in excitable tissues. The channels comprise a multisubunit complex consisting of a large (230-270kDa) highly glycosylated a subunit and one or two smaller subunits (pi and p2) (Goldin, 1994). The a subunits are part of a multigene family with at least nine members discovered so far in mammals (Mandel, 1992; Catterall, 1992; Fish et al., 1995; Klugbauer et al., 1995; Akopian et al., 1996; Sangameswaran et al., 1996; Toledo-Aral et al., 1997).

A potent blocker of VGSCs is the puffer fish toxin, tetrodotoxin (TTX). While most VGSCs are TTX-sensitive (TTX-s) and are inhibited by low nanomolar concentrations of TTX, there are two channels which are only inhibited by micromolar concentrations of TTX. These are the TTX resistant (TTX-r) major cardiac channel h1/SKM2 and the sensory neuron specific channel SNS/PN3 (Goldin, 1994; Akopian et al., 1996; Sangameswaran et al., 1996).

Primary sensory neurons express several distinct kinetic types of VGSCs. Small diameter neurons, most of which are high threshold nociceptors, co-express a rapidly inactivating, fast TTX sensitive current and a slowly activating and inactivating TTX-r sodium current. The larger diameter cells only express a TTX sensitive sodium current (Caffrey et al., 1992; Roy and Narahashi, 1992; Elliott and Eliott, 1993). Molecular distribution studies have suggested that these sodium currents are the result of the differential expression of different VGSCs, the TTX-s current being mediated by PN1, rSCP6/PN4, rBI, rBII or rBIII, which are found in large and small sensory neurons and the TTX-r current by SNS/PN3 which is found in small and medium-sized DRG neurons (Aguayo and White,

1992; Waxman et al., 1994; Black and Waxman, 1996; Black et al., 1996; England et al., 1996).

The TTX-r sodium current in the DRG is of interest because its differential expression in small neurons, predominately nociceptors (Akopian et al., 1996), offers the possibility of blocking activity only in these pain signalling afferents. The TTX-r current is, moreover, augmented by inflammatory mediators and may, therefore, contribute to the sensitisation of nociceptor terminals during inflammation (England et al., 1996; Gold et al., 1996). Following chronic constriction injury of the sciatic nerve, SNS/PN3 protein translocates from the cell body to the peripheral axons accumulating at the site of injury (Novakovic et al., 1998). Together, these data imply a key role for the TTX-r in the activation of nociceptors in inflammatory and neuropathic pain.

The TTX-r current recorded from isolated DRG neurons, appears to be relatively heterogeneous, displaying different kinetic properties in different neurons (Rizzo et al., 1994; Haper and Lawson, 1985; Elliott and Elliott, 1993; Rush and Elliott, 1997; Scholz et al., 1998). This raises the possibility of the existence of TTX-r molecular components other than SNS/PN3. Simon Tate and colleagues therefore attempted to find other TTX-r sodium channel a subunits expressed by primary sensory neurons. Using degenerate primers to domain IV of brain, skeletal muscle and glial sodium channels, they PCR amplified a band from rat genomic DNA that was used to screen a rat DRG cDNA library. A full length transcript was isolated that had 63% homology to SNS/PN3, 65% homology to h1/SKM2. Multiple tissue Northern blots indicated that expression was DRG specific, as observed for SNS/PN3 (Fig. 1). This putative sodium channel a subunit was therefore named SNS2. Deduced protein sequence revealed that SNS2 has a serine residue at the

site critical for TTX sensitivity (S-355) that should confer TTX resistance (Sivilotti et al., 1997). The expression of SNS2 in the DRG

and whether it colocalises with SNS/PN3 was studied using in situ

hybridisation and immunocytochemistry.

Figure 1; Northern blot showing the tissue distribution of SNS2 expression. A 7.5kb band was only ever observed in the total RNA prepared from the DRG.

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Methods

In situ hybridisation, immunocytochemistry and Northern blots were carried out as described in Chapter 4. Double labelling was done as

described in Chapter 5. Riboprobes were generated by in vitro

transcription, as described in Chapter 4, using a 1kb fragment of SNS/PN3 and a 1 kb fragment of SNS2 (corresponding to the second intracellular loop) in the PCR2.1 TA cloning vector (Invitrogen).

Results

SNS and SNS2 mRNA and protein are expressed by small sensory neurons

In situ hybridisation and immunohistochemistry showed SNS2 mRNA and protein labelling restricted to small diameter (10-25pm) DRG neurons (Fig. 2). Similar experiments performed for SNS/PN3 mRNA and protein in adjacent sections from the same ganglion showed labelling of small (10-25pm), and medium sized (25-40|am) neurons (Fig. 3). Neither SNS2 or SNS/PN3 staining was observed in non­ neuronal cells in the DRG. A size-frequency analysis of DRG cell profiles labelled for SNS/PN3 and SN2 mRNA illustrates clearly the difference in the size distribution of SNS2 expressing cells compared with SNS/PN3 positive cells (Figures 2 & 3). The same size distribution was found for immunolabelled SNS/PN3 and SNS2 neurons.

SNS and SNS2 are colocalised In small but not large sensory neurons

Double labelling for SNS/PN3 mRNA and SNS2 protein in the same section showed colocalisation only in small diameter neuronal cell bodies (Figure 4A,B). Colocalisation only in small neurons was also apparent when double labelling experiments were performed with SNS2 mRNA and SNS/PN3 protein (Figure 4 0 ,D). Large neurons were frequently seen with a signal for SNS/PN3 mRNA or protein where SNS2 protein or mRNA was absent.

Regulation of SNS2

SNS2 mRNA expression was not detectable in the DRG at embryonic day 15 (E l5), but was present on the day of birth (Figure

5). SNS/PN3, however, was detectable at E15, albeit at lower levels than postnatal day zero (PO). Neither SNS/PN3 nor SNS2 mRNA levels within lumbar DRGs showed any change in expression levels during postnatal development (between PO and adult), as determined by Northern blots probed separately with SNS2 followed by SNS/PN3 (Figure 5).

Both SNS/PN3 and SNS2 mRNA in the L4 and L5 DRG were down- regulated 48h and 7 days following sciatic nerve section, with the relative reduction in SNS2 more marked, as determined in the same Northern blots probed separately with SNS2 followed by SNS/PN3 (Figure 6). The mRNA expression for SNS2 remained unchanged in the L4 and L5 DRG at 24 and 48h following hind paw injection of complete Freund's adjuvant (CFA) to produce a local inflammation. At 7 days, post-inflammation, however, the SNS2 mRNA increased two-fold (Figure 6). The same Northerns probed for SNS/PN3 mRNA showed a general reduction after the inflammation, particularly at 48h.

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Profile Area (1xltf|inrf )

Figure 2: (A) In situ hybridisation and (B) immunocytochemistry for SNS2 in the DRG. Both the mRNA and the protein are localised in small DRG neurons (10-25|am). (C) Profile area / frequency histogram for SNS2 mRNA clearly shows that SNS2 expression is restricted to the very small DRG neurons.

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Figure 3: (A) In situ hybridisation and (B) immunocytochemistry for SNS/PN3 in the DRG. Both the

mRNA and the protein are localised in small DRG neurons (10-25pm) as well as medium sized (25-40|am) neurons. (C) Profile area / frequency histogram for SNS/PN3 mRNA shows that SNS2 expression is observed in small neurons, like SNS2, but also larger neurons, unlike SNS2.

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