In most neurons, including those mediating pain signals (nociceptive neurons), information, in the form of action potentials, is initiated through voltage-gated sodium channels (Nav). Local anaesthetics block pain signals through non-specific inhibition of Nav. Persistent pain may result from activation of distinct sensory nerve fibers in the pain pathway leading to acute, protective reflexes, or ill-adapted responses.
Researchers at the University of California, San Francisco sought to dissect out the specific contribution of different subtypes of Nav in mediating chemical, mechanical or thermal pain. Identification of selective Nav modulators from the venom of tarantula Heteroscodra maculate allowed scientists to address this goal in a study published in Nature on June 6. 1
Venoms from snakes, spiders and scorpions are complex in composition and include toxins which activate sensory nociceptors in predators, causing them pain or discomfort. Researchers screened venoms from 100 spiders, scorpions and centipedes using calcium (Ca2+) imaging to identify those activating mouse and rat somatosensory neurons in culture. They thus identified the venom from Heteroscodra maculate as a strong activator of trigeminal and dorsal root ganglia (DRG) neurons. Following venom fractionation, 2 active peptides were isolated, identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy (MALDI-TOF) and Edelman sequencing, and named δ-therapotoxin-Hm1a and b (Hm1a and Hm1b).
Hm1a (500 nM) triggered robust Ca2+ responses when applied to DRG neurons, and a synthetic version of the peptide was used in subsequent experiments. The sodium (Na+) channel blocker, tetrodotoxin (TTX, 500 nM) blocked Hm1a-evoked Ca2+ responses, implicating these channels in Hm1a-mediated response.
These results were further validated by electrophysiological studies, and the Nav1.1 subtype was identified as mediating Hm1a-induced activation of somatosensory neurons. Investigators further showed that Hm1a led to hyperexcitability of those neurons, with enhanced spike frequency (28.3 ± 8.4%; from 6.5 to 8.6 ms), and inhibition of Na+ current inactivation, leading to prolonged action potentials.
In situ hybridization experiments located Nav1.1 to sensory neurons of medium diameter, the majority of which (>75%) are myelinated, and co-localization studies indicated that these were primarily Aδ fibers.
In order to determine whether Hm1a elicited pain, investigators injected the toxin (5 µM) into hind paws of mice, intervention which immediately triggered robust nocifensive behavior, as assessed by licking and biting of injected paw (n=10, P<.001). Fibers co-expressing TRPV1 and Nav1.1 were excluded as being responsive to Hm1a, as intrathecal injection of capsaicin failed to ablate this nocifensive behavior in mice also injected with Hm1a (n=5). TRPV1-expressing fibers are sensitive to heat; researchers therefore tested heat sensitivity in animals, and found that intraplantar injection of Hm1a (500 nM) enhanced mechanical but not heat sensitivity.
Irritable Bowel Syndrome (IBS) patients develop chronic abdominal pain, and in particular, chronic mechanical hypersensitivity. Results from this tarantula toxin study clearly and specifically implicate Nav1.1 in mechanonociception, researchers therefore sought to determine whether this channel was expressed in the gut and whether it contributed to mechanical sensitization in a mouse model of chronic visceral hypersensitivity (CVH). Electrophysiological studies of ex vivo gut-nerve preparations from CVH and control mice showed that a subset of mechanosensitive colonic fibers express Nav1.1 channels, that baseline mechanosensory responses were elevated in CVH vs controls (6 out of 15, defined as≥15% increase over baseline; P < .01), that Hm1a enhanced neuronal activity induced by mechanostimulation in 36% of CVH fibers, and that Nav1.1 channels were overexpressed in 64% of CVH colonic DRG neurons, as indicated by enhanced electrical excitability upon toxin application.
These results implicate Nav1.1 in mechanical nociception, and in particular, in mechanical hypersensitivity observed in IBS, and provide an avenue for pharmacologic intervention through specific blockade of this channel.
1.Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain doi:10.1038/nature17976