Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels of the central and peripheral nervous system that regulate synaptic activity from both pre- and postsynaptic sites. Nicotine binding to brain nAChRs is thought to underlie the induction of behavioral addiction to nicotine, probably as a result of desensitizing/inhibitory effects. Here, another commonly abused drug, cocaine, is shown to selectively inhibit particular nAChR subtypes with a potency in the low micromolar range by interacting with separate sites associated with the α4 and β4 nAChR subunits. Chimeric receptor subunits and site-directed mutants were used to localize sequence determinants of cocaine affinity to: 1) a region of α4 located between residues 128 and 267 and 2) a site within the pore-lining M2 domain of β4 that includes the 13′ phenylalanine residue. The voltage dependence for inhibition associated with each site is consistent with these results. Analysis of the effects of incorporation of mutant and chimeric subunits also permitted identification of sequence elements important in receptor activation. For α3-containing receptors, a region or regions contained within the N-terminal extracellular domain of neuronal β subunits influence the time course of responses to acetylcholine. Conversely, the 13′ residue of the β4 subunit M2 region is important in determining acetylcholine potency, indicating a role for this residue in agonist binding/gating processes. In summary, the present work describes sequence elements critical in both cocaine inhibition and acetylcholine activation of nAChRs and indicates that nAChRs may provide a site of interaction for the effects of nicotine and cocaine in the nervous system.