Identification of a new amino acid residue capable of modulating agonist efficacy at the homomeric nicotinic acetylcholine receptor, alpha7.

Neuronal nicotinic receptors (nAChRs) have been implicated in pathology associated with neurological diseases and aberrant cognitive states such as addiction and schizophrenia. The design of subtype-specific cholinergic drugs is dependent on identification of key amino acids that play a significant role in determining subunit-specific agonist efficacy. 1,1-Dimethyl-4-phenylpiperazinium (DMPP) and a series of piperazium (PIP)-derived cholinergic agonists (1,1 dimethyl-4-acetylpiperizinium iodide, EthylPIP, PropylPIP, and ButylPIP) were used to identify a site (position 84) in homomeric neuronal nAChRs, which is a partial determinant of pharmacological specificity. In contrast to absolutely conserved amino acids within the nicotinic superfamily, the amino acid in position 84 can be polar or nonpolar. The addition of one methylene to PropylPIP to form ButylPIP eliminated channel activation of but not binding to the chick alpha7 homomeric nAChR (leucine in position 84). In rat alpha7 (glutamine in position 84), ButylPIP was an agonist. 1, 1-Dimethyl-4-phenylpiperazinium, a structural analog of ButylPIP, activates the rat alpha7 but is a weak partial agonist of the chick alpha7. Mutation of the chick alpha7 (L84Q) restored activation by ButylPIP, and the corresponding mutation in rat alpha7 (Q84L) abolished activation by ButylPIP. These mutations had moderate effects on the apparent affinity for acetylcholine, increasing its affinity for chick alpha7 and decreasing it for rat alpha7. Thus, the amino acid in position 84 (a residue on the periphery of the highly conserved loop A of the cys-loop superfamily of receptors) can potentially be exploited to produce subtype-specific drugs and can provide insights into the structure of the binding domain.

Subtype-selective inhibition of neuronal nicotinic acetylcholine receptors by cocaine is determined by the alpha4 and beta4 subunits.

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 alpha4 and beta4 nAChR subunits. Chimeric receptor subunits and site-directed mutants were used to localize sequence determinants of cocaine affinity to: 1) a region of alpha4 located between residues 128 and 267 and 2) a site within the pore-lining M2 domain of beta4 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 alpha3-containing receptors, a region or regions contained within the N-terminal extracellular domain of neuronal beta subunits influence the time course of responses to acetylcholine. Conversely, the 13' residue of the beta4 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.

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes.

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-microseconds time resolution, respectively, for studying neurotransmitter receptors in giant (approximately 20-microns diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.