Elucidating the role of NaV1.6 in peripheral sensory neurons

Abstract

Chronic pain is estimated to affect 1 in 5 Australians. The condition is debilitating for patients and remains a costly burden on the healthcare system. Analgesics currently in clinical use lack efficacy, have low tolerability and are hampered with wide side-effect profiles. Therefore, the underlying molecular mechanisms of pain sensing must be elucidated with a view to the development of improved therapeutics. Peripheral sensory neurons are the site of pain signal initiation and are critical for the detection of painful stimuli. Neuronal excitability relies on integral membrane bound proteins such as voltage-gated sodium channels (NaV), which allow the influx of sodium and action potential propagation in these cells. Nine different NaV subtypes have been identified (NaV1.1-1.9) of which a subset are expressed in peripheral sensory neurons. The exact role that each NaV isoform plays in the excitability of different fibre types remains unclear. This thesis uses a multidisciplinary approach to assess the role of the NaV subtype NaV1.6 in peripheral sensory neurons.NaV1.6 channels share high sequence homology with other NaV isoforms expressed in peripheral sensory neurons. Scorpion venom is a rich source of bioactive compounds, many of which act on mammalian NaV channels. One such peptide, Cn2, is reported to be a selective NaV1.6 activator. Chapter 2 describes the chemical synthesis of Cn2 using a strategy of native chemical ligation. The pharmacology of the synthetically derived Cn2 is in line with that of the venom derived peptide. This approach allows the development of a number of probes, including a novel NaV1.6 antagonist Cn2[E15R], that retain selectivity for NaV1.6. Therefore, this chapter characterises novel NaV1.6 selective compounds and provides insight into NaV1.6-toxin interactions.Peripheral sensory neurons express a range of different ion channels that are critical for their function. Chapter 3 aimed to assess the contribution of NaV1.6 to Na + currents in mouse isolated dorsal root ganglion cells. High-content calcium imaging and whole-cell patch clamp electrophysiology was performed using the selective Cn2 peptide as a probe for NaV1.6 function. Corresponding to expression of NaV1.6, Cn2 induces early Na + currents in large but not small diameter DRG neurons. The Cn2 effect is absent in NaV1.6 -/neurons confirming the selectivity of the peptide in a neuronal environment. Furthermore, Cn2 enhances excitability of large diameter neurons in vitro.