Dissecting the Inhibitory and Potentiation Effects of Desformylflustrabromine on a Muscle-Type Nicotinic Acetylcholine Receptor (NACHR)

Abstract

Desformylflustrabromine (dFBr) potentiates 4β2 nAChR in vitro (Kim et al. 2007, Biorg. Med. Chem. Lett. 17: 4855) and reduces nicotine self-administration in vivo (Liu 2013; Psychopharmacology 230: 203). We have reported that dFBr is a potent inhibitor (IC50, ∼1 M) of human (Hα12β1eδ) and Torpedo (Tα12β1γδ) muscle-type nAChRs. We also found, using [3H]dFBr reversible binding and dFBr displacement of [3H]PCP binding, that dFBr binds with high affinity to the muscle-type nAChRs ion channel in the desensitized state. But, when we photolabeled nAChR-rich Torpedo membranes with [3H]dFBr in the presence of agonist, four dFBr binding sites were evident. [3H]dFBr photolabeled amino acids in the ion channel and in three binding sites within the extracellular domain previously identified for nAChRs PAMs galanthamine and physostigmine. The high affinity binding of dFBr in the ion channel precludes the ability to study the functional contribution of dFBr binding within the extracellular domain and to determine whether dFBr acts as inhibitor or potentiator at these sites. Therefore, we are introducing mutations in the muscle-type nAChR ion channel M2 helices to eliminate the channel blocking effect of dFBr. The effect of M2 mutations on the EC50 of ACh and on dFBr inhibition of muscle-type nAChR were examined using two-electrode voltage clamp recording. Serine substitutions of the leucine at M2-9 of each nAChR subunit decreased dFBr IC50 compared with wild-type, consistent with an open channel blocking mechanism. This results confirm our [3H]dFBr the photolabeling results demonstrating that dFBr binds within the ion channel in close proximity to M2-9. The effects of other substitutions at M2-9 and at other positions in the M2 helices on dFBr inhibition of muscle-type nAChR are being tested.