Characterization of Human α4β2-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed in Native Nicotinic Receptor-Null SH-EP1 Human Epithelial Cells

Abstract

Naturally expressed nicotinic acetylcholine receptors composed of ␣4 and ␤2 subunits (␣4␤2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human ␣4␤2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human ␣4 and ␤2 subunits were stably introduced into the native nAChR-null human epithelial cell line SH-EP1. Heterologously expressed ␣4␤2-nAChR engage in high-affinity, specific binding of Rbϩ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-␤-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human ␣4␤2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human ␣4␤2-nAChR.

Cholinergic drugs potentiate human nicotinic α4β2 acetylcholine receptors by a competitive mechanism

“…Research into function and pharmacology of human a4h2 nicotinic acetylcholine receptors has been fostered by the availability of systems for the selective expression of specific subtypes of nicotinic acetylcholine receptors, such as Xenopus laevis oocytes Chavez-Noriega et al, 1997), human embryonic kidney (HEK) cells Buisson et al, 1996;Chavez-Noriega et al, 2000), and, more recently, human epithelial SH-EP1 cells (Eaton et al, 2003). Agonists of the nicotinic acetylcholine receptor interact with the agonist recognition sites of these receptors to induce conformational changes in the membrane protein, which lead to ion channel opening and receptor desensitization.…”

Rat neuronal nicotinic acetylcholine receptors containing α7 subunit: pharmacological properties of ligand binding and function

“…Stably transfected mammalian cell lines heterologously expressing neuronal nAChR subtypes have been powerful tools to study neuronal nAChRs [11,[27][28][29][30][31][32] . Several such cell lines expressing functional α7 nAChRs were successfully established in recent years, including the rat α7 subunit gene expressed in SH-SY5Y human neuroblastoma cells, which also express endogenous nAChR α3, α5, α7, β2, and β4 subunit genes [33] ; the human α7 subunit gene expressed in HEK-293 cells [11] and in SH-EP1 clonal human epithelial cells [34,35] , both of which are devoid of endogenous nAChR subunits; and the rat α7 subunit gene expressed in GH 4 C 1 clonal rat pituitary cells, which endogenously express the rat nAChR β4 subunit gene [36,37] .…”

Mouse β-TC6 Insulinoma Cells: High Expression of Functional α3β4 Nicotinic Receptors Mediating Membrane Potential, Intracellular Calcium, and Insulin Release

“…Human IMR-32 (Nelson et al, 2001) and human SH-SY5Y neuroblastoma cells (Dajas-Bailador et al, 2002), express a ganglionic subtype, but the subunit compositions are not known with certainty, because several subunits are expressed in these cells. Mammalian cells, stably transfected with different combinations of ␣ and ␤ nicotinic subunits, have proven useful for many studies (Whiting et al, 1991;Gopalakrishnan et al, 1996;Stauderman et al, 1998;Wang et al, 1998;Meyer et al, 2001;Eaton et al, 2003;Xiao and Kellar, 2004). However, the properties of receptors in transfected cells may not be fully equivalent to the properties in native systems because of ancillary proteins or other membrane components.…”