α4β2 Nicotinic Receptors with High and Low Acetylcholine Sensitivity: Pharmacology, Stoichiometry, and Sensitivity to Long-Term Exposure to Nicotine

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Abstract
α4 and β2 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 α4to β2 ratio. In this study, injection of oocytes with 1:10 α4/β2 subunit cDNA ratios favored expression of HS α4β2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS α4β2 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 α4 and β2. The 1:10 injection ratio produced the (α4)2(β2)3 stoichiometry, whereas 10:1 injections produced the (α4)3(β2)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-β-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 α4β2 receptors assembled in human embryonic kidney 293 cells. We conclude that nicotine up-regulates HS α4β2 receptors, probably by influencing the assembly of receptors rather than by altering the functional state of LS α4β2 nAChRs.