alpha 4 beta 2 nicotinic receptors with high and low acetylcholine sensitivity: Pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine

Alpha 4 and beta 2 nicotinic acetylcholine receptor ( nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the alpha 4 to beta 2 ratio. In this study, injection of oocytes with 1: 10 alpha 4/beta 2 subunit cDNA ratios favored expression of HS alpha 4 beta 2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10: 1 cDNA ratios favored expression of LS alpha 4 beta 2 receptors. The stoichiometry was inferred from the shifts in the ACh EC50 values caused by Leu to Thr mutations at position 9' of the second transmembrane domain of alpha 4 and beta 2. The 1: 10 injection ratio produced the (alpha 4)(2)(beta 2)(3) stoichiometry, whereas 10:1 injections produced the (alpha 4)(3)(beta(2))(2) stoichiometry. The agonists epibatidine, 3-[2(S)-azetidinylmethoxy]pyridine (A-85380), 5-ethoxy-metanicotine (TC-2559), cytisine, and 3-Br-cytisine and the antagonists dihydro-beta-erythroidine and d-tubocurarine were more potent at HS receptors. TC-2559 was more efficacious than ACh at HS receptors but was a partial agonist at LS receptors. Epibatidine was more efficacious than ACh at LS receptors and a partial agonist at HS receptors. Cytisine and 5-halogenated cytisines had moderate efficacy at LS receptors but had almost no efficacy at HS receptors. By exploiting the differential effects of ACh, TC-2559 and 5-I-cytisine we evaluated the effects of long-term exposure to nicotine on HS and LS receptors expressed in Xenopus laevis oocytes after cDNA injections or microtransplantation of alpha 4 beta 2 receptors assembled in human embryonic kidney 293 cells. We conclude that nicotine up-regulates HS alpha 4 beta 2 receptors, probably by influencing the assembly of receptors rather than by altering the functional state of LS alpha 4 beta 2 nAChRs