Charles W. Luetje

Varenicline Is a Partial Agonist at α4β2 and a Full Agonist at α7 Neuronal Nicotinic Receptors

Varenicline, a new nicotinic ligand based on the structure of cytisine, has recently been approved by the U.S. Food and Drug Administration for use as a smoking cessation aid. Varenicline has been shown to be a partial agonist of α4β2 receptors, and in equilibrium binding assays, it is highly selective for the α4β2 receptor. In this study, we have examined the functional activity of varenicline at a variety of rat neuronal nicotinic receptors expressed in Xenopus laevis oocytes and assayed under two-electrode voltage clamp. We also find that varenicline is a potent, partial agonist at α4β2 receptors, with an EC50 of 2.3 ± 0.3 μM and an efficacy (relative to acetylcholine) of 13.4 ± 0.4%. Varenicline has lower potency and higher efficacy at α3β4 receptors, with an EC50 of 55 ± 8 μM and an efficacy of 75 ± 6%. Varenicline also seems to be a weak partial agonist at α3β2 and α6-containing receptors, with an efficacy <10%. It is remarkable that varenicline is a potent, full agonist at α7 receptors with an EC50 of 18 ± 6 μM and an efficacy of 93 ± 7% (relative to acetylcholine). Thus, whereas varenicline is a partial agonist at some heteromeric neuronal nicotinic receptors, it is a full agonist at the homomeric α7 receptor. Some combination of these actions may be involved in the mechanism of varenicline as a smoking cessation aid.

Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors

Two properties were found to distinguish neuronal from muscle nicotinic acetylcholine receptors (nAChRs). First, neuronal nAChRs have a greater Ca2+ permeability. The high Ca2+ flux through neuronal nAChRs activates a Ca(2+)-dependent Cl- conductance, and the Ca2+ to Cs+ permeability ratio (PCa/PCs) is 7 times greater for neuronal than for muscle nAChRs. A second difference between the receptor types is that neuronal nAChRs are potently modulated by physiological levels of external Ca2+. Neuronal nAChR currents are enhanced by external Ca2+ in a dose-dependent manner. The results indicate that changes in extracellular Ca2+ modulate neuronal nAChRs and may modulate cholinergic synapses in the CNS. Also, activation of neuronal nAChRs produces a significant influx of Ca2+ that could be an important intracellular signal.

The postsynaptic 43k protein clusters muscle nicotinic acetylcholine receptors in xenopus oocytes

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.

Neurotoxins Distinguish Between Different Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations

Neuronal and muscle nicotinic acetylcholine receptor subunit combinations expressed in Xenopus oocytes were tested for sensitivity to various neurotoxins. Extensive blockade of the α3β2 neuronal subunit combination was achieved by 10 nM neuronal bungarotoxin. Partial blockade of the α4β2 neuronal and α1β1γδ muscle subunit combinations was caused by 1,000 nM neuronal bungarotoxin. The α2β2 neuronal subunit combination was insensitive to 1,000 nM neuronal bungarotoxin. Nearly complete blockade of all neuronal subunit combinations resulted from incubation with 2 nM neosurugatoxin, whereas 200 nM neosurugatoxin was required for partial blockade of the α1β1γδ muscle subunit combination. The α2β2 and α3β2 neuronal subunit combinations were partially blocked by 10,000 nM lophotoxin analog‐1, whereas complete blockade of the α4β2 neuronal and α1β1γδ muscle subunit combinations resulted from incubation with this concentration of lophotoxin analog‐1. The α1β1γδ muscle subunit combination was blocked by the α‐conotoxins G1A and M1 at concentrations of 100 nM. All of the neuronal subunit combinations were insensitive to 10,000 nM of both α‐conotoxins. Thus, neosurugatoxin and the α‐conotoxins distinguish between muscle and neuronal subunit combinations, whereas neuronal bungarotoxin and lophotoxin analog‐1 distinguish between different neuronal subunit combinations on the basis of differing α subunits.

Differential Inhibition by α‐Conotoxin‐MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices

Abstract: The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α‐conotoxin‐MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)‐anatoxin‐a‐evoked release of [3H]dopamine from rat striatal synaptosomes and slices. α‐Conotoxin‐MII (112 nM) completely blocked acetylcholine‐evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα‐conotoxin‐MII. On perfused striatal synaptosomes and slices, α‐conotoxin‐MII dose‐dependently inhibited [3H]dopamine release evoked by 1 µM (±)‐anatoxin‐a with IC50 values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose‐response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α‐conotoxin‐MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2‐like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)‐anatoxin‐a‐evoked [3H]dopamine release by α‐conotoxin‐MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α‐conotoxin‐MII‐insensitive nAChR.

Multiple Determinants of Dihydro-β-Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro‐β‐erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro‐β‐erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro‐β‐erythroidine by ∼120‐fold. IC50 values for blocking α4β4 and α3β4, responding to EC20 concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µM, respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro‐β‐erythroidine sensitivity to be distributed throughout the N‐terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro‐β‐erythroidine sensitivity by 4.3‐fold.

Nicotine receptors in the mammalian brain.

Nicotine is a drug of abuse that presumably exerts its psychoactive effect through its interactions with nicotine binding sites in the central nervous system. Among its potential sites of action are the neuronal nicotinic acetylcholine receptors and the neuronal α‐bungarotoxin binding sites. In this review we focus on the neuronal nicotinic acetylcholine receptors, their diversity, distribution, and functions as nicotine receptors or as mediators of synaptic transmission in the mammalian brain. We find that the complexity characteristic of the gene family encoding the subunits of these receptors is reflected both in the pattern of expression of the genes and in the pharmacological diversity of the expressed receptors.—Luetje, C. W.; Patrick, J.; Séguéla, P. Nicotine receptors in the mammalian brain. FASEB J. 4: 2753‐2760; 1990.

A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid

By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The “queen substance” [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queens dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50 = 280 ± 31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.

BIOMIMETIC CHEMICAL SENSORS USING NANOELECTRONIC READOUT OF OLFACTORY RECEPTORS

We have designed and implemented a practical nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells make them important pharmaceutical targets. Specifically, we have coupled olfactory receptor proteins (ORs) with carbon nanotube transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress on a path toward a bioelectronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.

Sex Pheromone Receptor Specificity in the European Corn Borer Moth, Ostrinia nubilalis

Background The European corn borer (ECB), Ostrinia nubilalis (Hubner), exists as two separate sex pheromone races. ECB(Z) females produce a 97∶3 blend of Z11- and E11-tetradecenyl acetate whereas ECB(E) females produce an opposite 1∶99 ratio of the Z and E isomers. Males of each race respond specifically to their conspecific females blend. A closely related species, the Asian corn borer (ACB), O. furnacalis, uses a 3∶2 blend of Z12- and E12-tetradecenyl acetate, and is believed to have evolved from an ECB-like ancestor. To further knowledge of the molecular mechanisms of pheromone detection and its evolution among closely related species we identified and characterized sex pheromone receptors from ECB(Z). Methodology Homology-dependent (degenerate PCR primers designed to conserved amino acid motifs) and homology-independent (pyrophosphate sequencing of antennal cDNA) approaches were used to identify candidate sex pheromone transcripts. Expression in male and female antennae was assayed by quantitative real-time PCR. Two-electrode voltage clamp electrophysiology was used to functionally characterize candidate receptors expressed in Xenopus oocytes. Conclusion We characterized five sex pheromone receptors, OnOrs1 and 3–6. Their transcripts were 14–100 times more abundant in male compared to female antennae. OnOr6 was highly selective for Z11-tetradecenyl acetate (EC50 = 0.86±0.27 µM) and was at least three orders of magnitude less responsive to E11-tetradecenyl acetate. Surprisingly, OnOr1, 3 and 5 responded to all four pheromones tested (Z11- and E11-tetradecenyl acetate, and Z12- and E12-tetradecenyl acetate) and to Z9-tetradecenyl acetate, a behavioral antagonist. OnOr1 was selective for E12-tetradecenyl acetate based on an efficacy that was at least 5-fold greater compared to the other four components. This combination of specifically- and broadly-responsive pheromone receptors corresponds to published results of sensory neuron activity in vivo. Receptors broadly-responsive to a class of pheromone components may provide a mechanism for variation in the male moth response that enables population level shifts in pheromone blend use.