Functional expression of nicotinic acetylcholine receptors containing rat α7 subunits in human SH‐SY5Y neuroblastoma cells

Abstract

Neuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned a7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes.Electrophysiological studies reveal that these all-nAChR function as a-bungarotoxin (Bgt)-sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt-sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional a7-nAChR in cell lines have failed. Here we report the successful use of SH-SYSY human neuroblastoma cells, which naturally express low levels of endogenous a7 transcripts, to stably overexpress heterologous rat nAChR a7 transgenes. These transgenes are expressed as the appropriately-sized a7 messages and protein, and stably transfected SH-SYSY cells have over 30-times higher levels of specific Bgt binding sites than do wild-type cells. Whole cell current recordings confhm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of a7-nAChR. We conclude that stable, functional expression of a7 transgenes in a mammalian cell line has been achieved for the first time.Key words: Acetylcholine; Nicotine; Nicotinic receptor; a-Bungarotoxin; SH-SYSY cell line lntroauctionNicotinic acetylcholine receptors (nAChR) exist as diverse members of the ligand-gated ion channel (LGIC) superfamily of neurotransmitter receptors [l-3]. Part of the diversity in nAChR is attributable to the existence of at least fifteen different nAChR subunit genes. Proteins encoded by these genes combine in different ways to generate several unique nAChR subtypes. However, roles of nAChR subunits, particularly in the formation and function of neuronal nAChR, are incompletely understood.NeuronaYnicotinic alpha-bungarotoxin binding sites (nBgtS) represent a unique class of nAChR that was first discovered in autonomic and CNS neurons or nervous tissue based on their ability to bind radiolabeled, curaremimetic neurotoxins such as a-bungarotoxin (Bgt) with high affiity and specificity [2,4-g]. Found where nAChR expression was expected, nBgtS also display a clearly nicotinic ligand binding profile, possess many of the physical and chemical properties of other nAChR, and have drug reactivities consistent with roles as LGIC. However, whereas studies of autonomic or CNS neurons that express nBgtS detected functional nAChR responses involved in the mediation of excitatory neurotransmission, those responses were not demonstrated to be sensitive to blockade by curaremimetic neurotoxins (although there were some exceptions; op. cit.). Thus, functional relevance of nBgtS and their identity as LGIC was questioned.Critical breakthroughs in our understanding of nBgtS ...

Functional Properties of Homomeric, Human α7-Nicotinic Acetylcholine Receptors Heterologously Expressed in the SH-EP1 Human Epithelial Cell Line

“…D, current-voltage curves showing peak current amplitudes ‫,ء(‬ normalized to the response in the same cell at Ϫ100 mV) as a function of holding potentials (abscissa; in millivolts) for recordings made from 6 to 12 cells and for data points as means Ϯ S.E.M. These values for channel closing are comparable to those observed the fast transient currents recorded from SH-SY5Y cells stably transfected with the rat ␣7 cDNA (7 ms; Puchacz et al, 1994a), and for ␣7-nAChR-mediated inward current responses reported in transfected ␣7-nAChR in the HEK-293 cell line (6 ms; Gopalakrishnan et al, 1995). Current findings also indicate that heterologously expressed human ␣7-nAChR-mediated peak current responses to nicotinic agonists applied at 3-min intervals and measured using K ϩ electrodes undergo "rundown", declining in amplitude with each successive application of agonist.…”

β-Amyloid Directly Inhibits Human α4β2-Nicotinic Acetylcholine Receptors Heterologously Expressed in Human SH-EP1 Cells

“…However, oocyteexpressed rat ␣7-nAChR responses are reported to be activated by the initial application of 1-100 pM A␤ (26). Other studies using acutely dissociated, rat basal forebrain neurons indicated ␤-Amyloid Inhibits Human ␣4␤2-nAChRsthat 4 M A␤ [25][26][27][28][29][30][31][32][33][34][35] or 100 nM A␤ 1-42 directly activated single channels and that 10 or 100 nM A␤ induced whole cell current responses attributable to a non-␣7-nAChR subtype (likely ␣4␤2-nAChR), also suggesting agonist-like activity of A␤ on nAChRs (43). Our observations showing (for the first time) significant functional inhibition of h␣4␤2-nAChRs by 1 nM A␤ and finding (consistent with cross-species observations) (22)(23)(24)(25)(26) inhibition of h␣7-nAChRs by 100 nM A␤ indicate that A␤ 1-42 acts as a nAChR functional antagonist with higher affinity for h␣4␤2-nAChRs than for h␣7-nAChRs.…”

“…Heterologously Expressed h␣4␤2-nAChRs in SH-EP1 Cells-Human ␣4 and ␤2 subunits (kindly provided by Dr. Ortrud Steinlein, Institute for Human Genetics, Rheinische-Wilhelms-Universitat, Bonn, Germany) were subcloned into pcDNA3.1-zeocin and pcDNA3.1-hygromycin vectors, respectively, and transfected using established techniques (32)(33)(34) into native nAChR-null SH-EP1 cells (35) to create the SH-EP1-h␣4␤2 cell line. For this and all other methods, manipulations were conducted at room temperature (23 Ϯ 1°C) unless otherwise noted.…”

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

“…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] .…”