Characterization of Human α4β2 Neuronal Nicotinic Receptors Stably Expressed in SH-EP1 Cells

Abstract

These studies characterized human α4β2 neuronal nicotinic receptors stably expressed in a human epithelial cell line (SH-EP1). Receptors in transfected SH-EP1-hα4β2 cells were functional, as determined by increases in intracellular Ca²⁺ in response to a nicotine stimulus. Nicotine increased Fura-2 fluorescence in a concentration-dependent manner with an apparent EC₅₀ of 2.4 μM, a response that was blocked by the specific antagonist mecamylamine. When cells were incubated in 50 nM nicotine for 24 hours, the Ca²⁺ response inactivated by 44%, an effect that recovered within 24 hours. SH-EP1-hα4β2 cells expressed a single class of high affinity binding sites for [³H]cytisine with a Kd of 0.63 ± 0.08 nM and a Bmax of 6797 ± 732 femtomoles/mg protein. Incubation of cells with 50 nM nicotine for 24 hours increased the Bmax by 45% without changing affinity, a concentration-dependent effect with an EC₅₀ of 58.6 nM. The nicotine-induced up regulation was reversible, and control values were achieved within 24 hours. Results indicate that SH-EP1-hα4β2 cells may be a good model system to study regulation of human α4β2 receptors, the most abundant nicotinic receptor subtype in brain.