AChBP-targeted α-conotoxin correlates distinct binding orientations with nAChR subtype selectivity

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases

Characterization of a novel alpha-conotoxin TxID from Conus textile that potently blocks rat alpha3/beta4 nicotinic acetylcholine receptors

The alpha 3 beta 4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an alpha-4/6-conotoxin (CTx) TxID from Conus textile. The new toxin consists of 15 amino acid residues with two disulfide bonds. TxID was synthesized using solid phase methods, and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxID blocked rat alpha 3 beta 4 nAChRs with a 12.5 nM IC50, which places it among the most potent alpha 3 beta 4 nAChR antagonists. TxID also blocked the closely related alpha 6/alpha 3 beta 4 with a 94 nM IC50 but showed little activity on other nAChR subtypes. NMR analysis showed that two major structural isomers exist in solution, one of which adopts a regular alpha-CTx fold but with different surface charge distribution to other 4/6 family members. alpha-CTx TxID is a novel tool with which to probe the structure and function of alpha 3 beta 4 nAChRs

A novel mechanism of inhibition of high-voltage activated calcium channels by a-conotoxins contributes to relief of nerve injury-induced neuropathic pain

alpha-Conotoxins that are thought to act as antagonists of nicotinic acetylcholine receptors (nAChRs) containing alpha 3-subunits are efficacious in several preclinical models of chronic pain. Potent interactions of Vc1.1 with other targets have suggested that the pain-relieving actions of alpha-conotoxins might be mediated by either alpha 9 alpha 10 nAChRs or a novel GABA(B) receptor-mediated inhibition of N-type calcium channels. Here we establish that three alpha-conotoxins, Vc1.1, AuIB and MII have distinct selectivity profiles for these three potential targets. Their potencies after intramuscular administration were then determined for reversal of allodynia produced by partial nerve ligation in rats. Vc1.1, which potently inhibits alpha 9 alpha 10 nAChRs and GABA(B)/Ca(2+) channels but weakly blocks alpha 3 beta 2 and alpha 3 beta 4 nAChRs, produced potent, long-lasting reversal of allodynia that were prevented by pre-treatment with the GABA(B) receptor antagonist, SCH50911. alpha-Conotoxin AuIB, a weak alpha 3 beta 4 nAChR antagonist, inhibited GABA(B)/Ca(2+) channels but did not act on alpha 9 alpha 10 nAChRs. AuIB also produced reversal of allodynia. These findings suggest that GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels can mediate the sustained anti-allodynic actions of some alpha-conotoxins. However, MII, a potent alpha 3 beta 2 nAChR antagonist but inactive on alpha 9 alpha 10 and alpha 3 beta 4 nAChRs and GABA(B)/Ca(2+) channels, was demonstrated to have short-acting anti-allodynic action. This suggests that alpha 3 beta 2 nAChRs may also contribute to reversal of allodynia. Together, these findings suggest that inhibition of alpha 9 alpha 10 nAChR is neither necessary nor sufficient for relief of allodynia and establish that alpha-conotoxins selective for GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels relieve allodynia, and could therefore be developed to manage chronic pain. (C) 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved

Molecular mechanism for analgesia involving specific antagonism of α9α10 nicotinic acetylcholine receptors

α9α10 nicotinic acetylcholine receptors (nAChRs) have been identified in a variety of tissues including lymphocytes and dorsal root ganglia; except in the case of the auditory system, the function of α9α10 nAChRs is not known. Here we show that selective block (rather than stimulation) of α9α10 nAChRs is analgesic in an animal model of nerve injury pain. In addition, blockade of this nAChR subtype reduces the number of choline acetyltransferase-positive cells, macrophages, and lymphocytes at the site of injury. Chronic neuropathic pain is estimated to affect up to 8% of the world's population; the numerous analgesic compounds currently available are largely ineffective and act through a small number of pharmacological mechanisms. Our findings not only suggest a molecular mechanism for the treatment of neuropathic pain but also demonstrate the involvement of α9α10 nAChRs in the pathophysiology of peripheral nerve injury