Role of Voltage-gated Sodium Channel Isoforms in Electrophysiological Properties of Neurons Innervating the Viscera in Mice

Chronic visceral pain is a poorly managed symptom of functional and inflammatory gastrointestinal disorders and there is a lack of suitable analgesics that are efficacious without gastrointestinal side effects. Voltage-gated sodium (Nav) channels regulate action potential generation and cell membrane excitability in sensory neurons and are implicated in several enhanced pain and loss-of-pain phenotypes in humans. Pharmacological modulation of Nav channels has been investigated as a therapeutic strategy for the past two decades in a range of pain modalities, including somatic, neuropathic, and more recently - visceral pain. In this thesis, gene transcripts for the nine Nav channel isoforms (Nav1.1-Nav1.9) were detected in dorsal root ganglia (DRG) neurons retrogradely labeled from the colon and bladder in mice, and the contribution of different isoforms to active electrophysiological properties in these neurons was evaluated using Nav-selective modulators. An evaluation of electrophysiological properties of colon-innervating DRG neurons from healthy and chronic visceral hypersensitivity (CVH) mice was also conducted and did not provide sufficient support for a model-related phenotype in vitro. In the pharmacological part of this thesis, it was found that inhibition of tetrodotoxin-sensitive Nav channels (Nav1.1-Nav1.4, Nav1.6 and Nav1.7) effectively altered electrophysiological responses in colon-innervating and bladder-innervating neurons, and furthermore reduced bladder afferent responses to distension and nociceptive signaling to the spinal cord. Electrophysiological responses in colon-innervating DRG neurons were also modulated by less selective Nav modulators, such as veratridine, which targets all Nav channel isoforms, and more selective Nav modulators, such as Hs1a, which targets Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7; OD1, which targets Nav1.4, Nav1.6, and Nav1.7; ICA-121341, which targets Nav1.1-Nav1.3; A-803467, which targets Nav1.8; and Compound B, which targets Nav1.1. inhibition of Nav1.1 using Compound B was furthermore shown to be effective in reducing pain responses to colorectal distension in CVH mice.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation

Contribution of membrane receptor signalling to chronic visceral pain

Irritable bowel syndrome and inflammatory bowel disease are major forms of chronic visceral pain, which affect over 15% of the global population. In order to identify new therapies, it is important to understand the underlying causes of chronic visceral pain. This review provides recent evidence demonstrating that inflammation or infection of the gastrointestinal tract triggers specific changes in the neuronal excitability of sensory pathways responsible for the transmission of nociceptive information from the periphery to the central nervous system. Specific changes in the expression and function of a variety of ion channels and receptors have been documented in inflammatory and chronic visceral pain conditions relevant to irritable bowel syndrome and inflammatory bowel disease. An increase in pro-nociceptive mechanisms enhances peripheral drive from the viscera and provides an underlying basis for enhanced nociceptive signalling during chronic visceral pain states. Recent evidence also highlights increases in anti-nociceptive mechanisms in models of chronic visceral pain, which present novel targets for pharmacological treatment of this condition

Identification and characterization of ProTx-III [μ-TRTX-Tp1a], a new voltage-gated sodium channel inhibitor from venom of the tarantula Thrixopelma pruriens

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (Na-V) channels, we screened spider venoms for inhibitors of human Na(V)1.7 (hNa(V)1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na(V)1.7 inhibitor, mu-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa(V)1.7 > hNa(V)1.6 > hNa(V)1.2 > hNa(V)1.1 > hNa(V)1.3 channels in fluorescent assays. Na(V)1.7 inhibition was diminished (IC50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form(IC50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNa(V)1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 mu M. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNa(V)1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa(V)1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa(V)1.7 inhibitors for treatment of chronic pain