αS-conotoxin GVIIIB potently and selectively blocks α9α10 nicotinic acetylcholine receptors

Abstract

Although acetylcholine is widely utilized in vertebrate nervous systems, nicotinic acetylcholine receptors (nAChRs), including the α9α10 subtype, also are expressed in a wide variety of non-neuronal cells. These cell types include cochlear hair cells, adrenal chromaffin cells and immune cells. α9α10 nAChRs present in these cells may respectively play roles in protection from noise-induced hearing loss, response to stress and neuroprotection. Despite these critical functions, there are few available selective ligands to confirm mechanistic hypothesis regarding the role of α9α10 nAChRs. Conus, has been a rich source of ligands for receptors and ion channels. Here, we identified Conus geographus venom as a lead source for a novel α9α10 antagonist. The active component was isolated and the encoding gene cloned. The peptide signal sequence and cysteine arrangement had the signature of the σ-conotoxin superfamily. Previously isolated σ-conotoxin GVIIIA, also from Conus geographus, targets the 5-HT3 receptor. In contrast, αS-GVIIIB blocked the α9α10 nAChR with an IC50 of 9.8nM, yet was inactive at the 5-HT3 receptor. Pharmacological characterization of αS-GVIIIB shows that it is over 100-fold selective for the α9α10 nAChR compared to other nAChR subtypes. Thus, the S-superfamily represents a novel conotoxin scaffold for flexibly targeting a variety of receptor subtypes. Functional competition studies utilized distinct off-rate kinetics of conotoxins to identify the α10/α9 nAChR interface as the site of αS-GVIIIB binding; this adds to the importance of the (+) face of the α10 rather than the (+) face of the α9 nAChR subunit as critical to binding of α9α10-targeted conotoxins.