Agonist and antagonist effects of nicotine on chick neuronal nicotinic receptors are defined by alpha and beta subunits.
1. Functional neuronal nicotinic receptors were reconstituted in Xenopus oocytes by the nuclear injection of different combinations of chick and rat cDNAs encoding alpha and beta subunits. The pharmacology of these nicotinic receptors was investigated using two-electrode voltage clamp. 2. The sensitivity of the chick alpha 3/beta 2, alpha 3/beta 4, and alpha 4/beta 2 receptors to acetylcholine (ACh) and neuronal bungarotoxin differed markedly, indicating that both subunits contribute to the pharmacological properties of the receptors. 3. Nicotine acted as an agonist on the chick alpha 3/beta 4 and alpha 4/beta 2 receptors and rat alpha 3/beta 2 receptor. In contrast, nicotine (at concentrations > 3 microM) was only a weak partial agonist of the chick alpha 3/beta 2 receptor. Moreover, nicotine coapplied with 3 microM ACh on the chick alpha 3/beta 2 receptor acted as a potent competitive antagonist, with an IC50 of 0.43 microM. No antagonist effect of nicotine could be revealed on the other nicotinic receptors. 4. The effect of nicotine was tested on hybrid receptors obtained by coinjection of chick and rat cDNAs encoding the alpha 3 and beta 2 subunits (yielding the rat alpha 3/chick beta 2 and chick alpha 3/rat beta 2 receptors). Nicotine (10 microM) strongly inhibited both hybrid receptors. 5. Chimeric subunits were constructed by exchanging a segment located in the extracellular N-termini of chick alpha 3 and alpha 4 subunits and chick alpha 3 and rat alpha 3 subunits. These subunits were coexpressed in oocytes with chick or rat beta 2 subunits. The effect of nicotine on these receptors pointed to the importance of a 15 amino acid stretch located 3' of the first transmembrane segment in the determination of the agonist and antagonist action of nicotine. 6. Within this 15 amino acid segment, a single residue differs in chick and rat alpha 3 subunits, at position 198, within the ligand binding site of alpha subunits. Gln198 of the rat alpha 3 subunit was replaced by Thr as found in the chick alpha 3 subunit, using site-directed mutagenesis. The mutant subunit was coexpressed with the rat beta 2 subunit, yielding receptors which were inhibited by nicotine. 7. It is concluded that both alpha and beta subunits not only alter considerably the sensitivity of the nicotinic receptor to agonists and antagonists, but also can turn an agonist into a potent antagonist of one receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)
Hussy
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Département de Physiologie, Centre Médical Universitaire, Université de Genève, Switzerland.
Ballivet
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Bertrand
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D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009865:Oocytes; D051381:Rats; D011978:Receptors, Nicotinic / Q000145:classification* / Q000235:genetics / Q000502:physiology; D009435:Synaptic Transmission / Q000187:drug effects* / Q000502:physiology; D014981:Xenopus
D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D009538:Nicotine; D000109:Acetylcholine