RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia

Abstract

Transcripts for α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are found in diverse tissues. The function of α9α10 nAChRs is best known in mechanosensory cochlear hair cells, but elsewhere their roles are less well-understood. α9α10 nAChRs have been implicated as analgesic targets and α-conotoxins that block α9α10 nAChRs produce analgesia. However, some of these peptides show large potency differences between species. Additionally several studies have indicated that these conotoxins may also activate GABAB receptors (GABABRs). To further address these issues, we cloned the cDNAs of mouse α9 and α10 nAChR subunits. When heterologously expressed in Xenopus oocytes, the resulting α9α10 nAChRs had the expected pharmacology of being activated by acetylcholine and choline but not by nicotine. A conotoxin analog, RgIA4, potently, and selectively blocked mouse α9α10 nAChRs with low nanomolar affinity indicating that RgIA4 may be effectively used to study murine α9α10 nAChR function. Previous reports indicated that RgIA4 attenuates chemotherapy-induced cold allodynia. Here we demonstrate that RgIA4 analgesic effects following oxaliplatin treatment are sustained for 21 days after last RgIA4 administration indicating that RgIA4 may provide enduring protection against nerve damage. RgIA4 lacks activity at GABAB receptors; a bioluminescence resonance energy transfer assay was used to demonstrate that two other analgesic α-conotoxins, Vc1.1 and AuIB, also do not activate GABABRs expressed in HEK cells. Together these findings further support the targeting of α9α10 nAChRs in the treatment of pain.

Nicotinic acetylcholine receptors in neuropathic and inflammatory pain

“…One of these analogs, RgIA4, showed a~300-fold increase in potency and, critically, was > 1000-fold more potent on a9a10 nAChRs than a7 nAChRs. As previously discussed, RgIA4 has been shown to be an effective analgesic and prophylactic in the oxaliplatin model of neuropathic pain [112,143]. Additional conopeptides have also been recently discovered that selectively target a9-containing nAChRs [160,161].…”

“…Functionally, a9* nAChRs have been shown to modulate the release of IL-1b from human monocyte-derived U937 cells as well as from mouse peripheral blood mononuclear leukocytes [83, 135,136]. It is important to point out that in these studies nicotine functioned as an agonist in contrast to the antagonist action observed for oocyte-and hair cell-expressed a9* nAChRs [6, 112,123]. Furthermore, unlike oocyteand hair cell-expressed a9* nAChRs, those found in U937 cells and leukocytes do not function as canonical ligand-gated ion channels; application of agonists that activate oocyte-or hair cell-expressed a9-containing nAChRs do not evoke ion currents in tested immune cells.…”

RgIA4 Accelerates Recovery from Paclitaxel-Induced Neuropathic Pain in Rats

“…Neither paclitaxel nor RgIA4 affected heat allodynia, as measured by the Hargreaves test, nor the weight gain of the animals over the course of the experiment (Figure 4). The dosages of 80 and 16 µg/kg were chosen based on previously characterized regimens that effectively produced relief in neuropathic pain models in mice and rats [24,25]. These dosages have been previously reported to produce no adverse effects in motor coordination nor CNS function based on rotarod and Irwin tests [25].…”

“…RgIA4 has been previously shown to prevent the induction of CIPN pain by the platinum-based chemotherapeutic, oxaliplatin [24,25]. Since chemotherapeutic agents work by different mechanisms of action to inhibit tumor growth, we wished to assess the activity of RgIA4 in taxane-induced neuropathic pain [39].…”

“…This 13-amino-acid peptide, isolated from the worm-hunting snail Conus regius, blocked the rodent α9α10 nAChR with high potency; however, it was found to be approximately 300-fold less potent on the human receptor due to a single amino-acid substitution in the α9 nAChR subunit [23]. The secondgeneration synthetic analog, RgIA4, was engineered to close this affinity gap across the rodent and human α9α10 nAChRs (IC 50 rat = 0.9 nM; IC 50 human = 1.5 nM), and has also been shown to effectively prevent oxaliplatin-induced pain in rats and mice [24,25]. These findings are consistent with previous behavioral and cellular studies that demonstrate native RgIA can prevent the development and/or progression of neuropathic pain in chronic constriction injury and oxaliplatin-induced injury [26][27][28].…”