Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells

• Published in: Brain research Vol. 1542; pp. 1 - 11
• Main Authors: Rasmussen, Andreas H, Strøbæk, Dorte, Dyhring, Tino, Jensen, Marianne L, Peters, Dan, Grunnet, Morten, Timmermann, Daniel B, Ahring, Philip K
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 13.01.2014; Elsevier
• Subjects: Acetylcholine; Acetylcholine - metabolism; Acetylcholine - pharmacology; Alpha6; Beta2; Biological and medical sciences; Biophysical Phenomena - drug effects; Biophysical Phenomena - physiology; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Calcium Channel Blockers - pharmacology; Cell receptors; Cell structures and functions; Cholinergic Agents - pharmacology; Conotoxins - pharmacology; Dose-Response Relationship, Drug; Electrophysiology; Fundamental and applied biological sciences. Psychology; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Membrane Potentials - drug effects; Membrane Potentials - genetics; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Mutation - genetics; Neurology; Nicotine - pharmacology; Nicotinic; Nicotinic Agonists - pharmacology; Patch-Clamp Techniques; Plant Lectins - pharmacology; Protein Subunits - genetics; Protein Subunits - metabolism; Pyridines - pharmacology; Receptors; Receptors, Nicotinic - genetics; Receptors, Nicotinic - metabolism; Transfection; transmembrane segment; α6; ACh; Electrophysiology; α6-containing; Alpha6; Nicotinic; Receptors; Beta2; nAChR; TM; Acetylcholine; nicotinic acetylcholine receptor; Characterization; Cholinergic receptor; Treatment; Neurotransmitter; Nicotinic receptor; Biological receptor; α6(⁎)
• DOI: 10.1016/j.brainres.2013.10.024
• ISSN: 0006-8993

Abstract Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6⁎ ) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6⁎ receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6⁎ receptor expression. Combinations of chimeric and mutated α6, β2 and β3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca2+ -imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3V9'S gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3V9'S :β2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type β3, significant functional expression of α6/α3β2β3 was also demonstrated. Comparing the acetylcholine concentration–response relationship of α6/α3β2β3V9'S to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca2+ -imaging experiments showed preservation of basic α6⁎ receptor pharmacology. Our results establish that α6/α3β2β3V9'S replicate several basic features of native α6⁎ receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts.