5-I A-85380 and TC-2559 differentially activate heterologously expressed α4β2 nicotinic receptors

The neuronal nicotinic acetylcholine receptor α4 and β2 subunits expressed in heterologous expression systems assemble into at least two distinct subunit stoichiometries of α4β2 receptor. The (α4)2(β2)3 stoichiometry is about 100-fold more sensitive to acetylcholine than the (α4)3(β2)2 stoichiometry. In order to investigate if agonists in general distinguish high- and low-affinity α4β2 nicotinic acetylcholine receptors, we have expressed human α4 and β2 nicotinic acetylcholine receptor subunits in two different expression systems. The relative amounts of α4β2 nicotinic acetylcholine receptors with high- and low-affinity for acetylcholine were manipulated by (a) injecting the subunit cDNAs at different α:β ratios into Xenopus oocytes and (b) by culturing HEK-293 cells stably expressing α4β2 nicotinic acetylcholine receptors overnight at different temperatures. The sensitivities of the α4β2 nicotinic acetylcholine receptors to the agonists acetylcholine, 5-I A-85380, and TC-2559 were investigated using the voltage-clamp technique on Xenopus oocytes and using a fluorescent imaging plate reader to measure calcium responses from HEK-293 cells. Like acetylcholine, 5-I A-85380 produced biphasic concentration–response curves and the high-affinity component became larger when the cells were manipulated to produce a greater proportion of (α4)2(β2)3 nicotinic acetylcholine receptors. Interestingly, under all circumstances, TC-2559 produced monophasic concentration–response curves. In oocytes injected with α4 and β2 subunits in the 1:1 ratio the maximum effect of TC-2559 was 28% of that of acetylcholine. The EC50 for TC-2559 was not changed when oocytes were manipulated to express exclusively (α4)2(β2)3 nicotinic acetylcholine receptors, however, the maximum effect of TC-2559 was dramatically enhanced. These results suggest that TC-2559 is a selective agonist of the (α4)2(β2)3 nicotinic acetylcholine receptor stoichiometry.