Quek_2010_ACS.Chem.Neurosci_1_796

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the alpha7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat alpha7, alpha4beta2, and alpha3beta4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 muM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the alpha7 nAChR. At alpha4beta2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of alpha4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of alpha4beta2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (alpha4)(3)(beta2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.