Abstract

Ever since the discovery that mutations in the voltage-gated sodium channel 1.7 protein are responsible for human congenital insensitivity to pain, the voltage-gated sodium channel (NaV) family of ion channels has been the subject of intense research with the hope of discovering novel analgesics. We now know that NaV1.7 deletion in select neuronal populations yield different phenotypes, with the deletion of NaV1.7 in all sensory neurons being successful at abolishing mechanical and heat-induced pain. However, it is rapidly becoming apparent that a number of pain syndromes are not modulated by NaV1.7 at all, such as oxaliplatin-mediated neuropathy. It is particularly interesting to note that the loss of NaV1.7 function is also associated with the selective inhibition of pain mediated by specific stimuli, such as that observed in burn-induced pain where NaV1.7 gene knockout abolished thermal allodynia but did not affect mechanical allodynia. Accordingly, significant interests exist in delineating the contribution of other NaV isoforms in modality-specific pain pathways. Two other isoforms, NaV1.6 and NaV1.8, are now specifically implicated in some NaV1.7-independent conditions such as oxaliplatin-induced cold allodynia. Such selective contributions of specific ion channel isoforms to pain highlight the need to discover other putative protein targets involved in mediating nociception. The aim of my work is therefore to discover selective molecular inhibitors of NaV1.6 and NaV1.8, to find useful cell models for peripheral nociceptors, to investigate the roles of NaV1.6 and NaV1.8 in an animal model of burn-induced pain, and to screen for putative new targets for analgesia in burn-related pain. Chapter 2 of this thesis describes activity-guided discovery of novel NaV1.6 and NaV1.8-modulting peptides from crude spider venoms. Crude venom from Poecilotheria metallica successfully reduced NaV1.8-mediated voltage changes in HEK293-expressing cells. A new NaV1.8-inhibitory peptide was sequenced from the venom and named Pme1a. However, Pme1a exhibited TRPV1 agonist activity and induced nocifensive behaviours, such as paw licking, after injection into the hind paws of mice, indicating the peptide was not a selective NaV1.8 modulator and was unsuitable as a tool for NaV1.8 investigation. With the unsuccessful exploration of spider venoms for new specific inhibitors, I then investigated in vitro cell models as tools to research specific nociceptor subtypes that express NaV1.6 or NaV1.8. In Chapter 3, I provide the first complete transcriptome of three common in vitro neuronal cell lines (SH-SY5Y, F-11, and ND7/23) to examine whether they were appropriate for investigating the functions of NaV1.6 and NaV1.8, and to identify if the cell lines resemble in vivo dorsal root ganglion (DRG) neuronal subclasses. The three cell lines examined all expressed proteins belonging to similar pathways and of similar proportions to native DRG neurons. However, they did not express any cellular markers in a fashion that represented any known subclasses of DRG neurons. Therefore, it is unlikely that any of the cell lines examined are appropriate models for NaV1.6 or NaV1.8 function, and highlights the need for careful selection of models in in vitro work. As investigations for selective NaV1.6 and NaV1.8 modulators and appropriate in vitro cell models both proved to be unsuccessful, I then turned to animal models to study modality-specific contributions of NaV isoforms. Chapter 4 describes the establishment of a murine model of peripheral burn injury to assess the involvement of NaV1.6 and NaV1.8 in burn-induced pain. The inhibition of NaV1.6 and no other NaV isoforms significantly reduced burn-induced mechanical allodynia, a NaV1.7-independent pain modality, indicating NaV1.6 plays a vital role in this NaV1.7-independent condition. However, immunofluorescence studies revealed NaV1.6 expression was not changed in the affected DRGs. I therefore decided to conduct a transcriptomic investigation to examine whether the expression of other pain-related ion channel gene were changed, and to discover if genes differentially expressed in the affected DRG neurons could be potential pharmacological targets for analgesia. The results of the transcriptomic investigation of DRGs affected by the burn model are discussed in Chapter 5. A total of 30 genes were found to be differentially expressed, including genes known to be involved in nociception (such as neuropeptide Y) as well as genes with no known association to nociception (such as lipase, family member N). It should be noted that none of the NaV isoforms were differentially expressed, indicating that NaV channels may alter pain sensitivity through modulated function rather than expression. Selective inhibitors of the protein products of the genes found to be up-regulated were then tested in the burn-induced pain mouse model. Proglumide, an inhibitor of the cholecystokinin B receptor, exhibited a significant anti-allodynic effect in burn-induced mechanical allodynia and was synergistic with oxycodone. Some genes found to be differentially expressed in burn injury were also identified in the cell lines covered in Chapter 3, and the cell lines can potentially be used as in vitro models to rapidly profile the effects of inhibitors of these proteins in peripheral neurons.

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