AGONIST AND ANTAGONIST EFFECTS OF NICOTINE ON CHICK NEURONAL NICOTINIC RECEPTORS ARE DEFINED BY ALPHA-SUBUNITS AND BETA-SUBUNITS

1. Functional neuronal nicotinic receptors were reconstituted in Xenop us oocytes by the nuclear injection of different combinations of chick and rat cDNAs encoding alpha and beta subunits. The pharmacology of t hese 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 acetylchofne (ACh) and neuronal bunga rotoxin 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 a nd rat alpha 3/beta 2 receptor. In contrast, nicotine (at concentratio ns >3 mu M) was only a weak partial agonist of the chick (alpha 3/beta 2 receptor. Moreover, nicotine coapplied with 3 mu M ACh on the chick alpha 3/beta 2 receptor acted as a potent competitive antagonist, wit h an IC50 of 0.43 mu M. No antagonist effect of nicotine could be reve aled on the other nicotinic receptors. 4. The effect of nicotine was t ested on hybrid receptors obtained by coinjection of chick and rat cDN As 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 mu M) strongly inhibited both hybrid receptors. 5. Chimeric subunits were constructed by exchanging a segment located in the extracellular N-te rmini of chick alpha 3 and alpha 4 subunits and chick alpha 3 and rat alpha 3 subunits. These subunits were coexpressed in oocytes with chic k or rat beta 2 subunits. The effect of nicotine on these receptors po inted to the importance of a 15 amino acid stretch located 3' of the f irst transmembrane segment in the determination of the agonist and ant agonist action of nicotine. 6. Within this 15 amino acid segment, a si ngle residue differs in chick and rat alpha 3 subunits, at position 19 8, within the ligand binding site of a subunits. Gin 198 of the rat al pha 3 subunit was replaced by Thr as found in the chick alpha 3 subuni t, 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 a gonists and antagonists, but also can turn an agonist into a potent an tagonist of one receptor subtype. The antagonist action of nicotine pr obably depends on complex interactions between subunits, in which resi due 198 of alpha subunits plays a crucial role. The inhibitory effect of nicotine on subtypes of neuronal nicotinic receptors may have impor tant implications regarding the effect of nicotine in the brain.