title: Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells.
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 Ca(2+)-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3(V9'S) gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3(V9'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/α3β2β3(V9'S) to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca(2+)-imaging experiments showed preservation of basic α6 receptor pharmacology. Our results establish that α6/α3β2β3(V9'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.
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Rasmussen
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Dorte
authors.initials: D
authors.lastname: Strøbæk
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Tino
authors.initials: T
authors.lastname: Dyhring
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Marianne L
authors.initials: ML
authors.lastname: Jensen
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Dan
authors.initials: D
authors.lastname: Peters
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Grunnet
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Timmermann
authors.affiliation: Aniona ApS, Baltorpvej 154, DK-2750 Ballerup, Denmark. Electronic address: pka@aniona.com.
authors.forename: Philip K
authors.initials: PK
authors.lastname: Ahring
mesh_terms: D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D055592:Biophysical Phenomena / Q000187:drug effects* / Q000502:physiology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D018678:Cholinergic Agents / Q000494:pharmacology*; D020916:Conotoxins / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D037121:Plant Lectins / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D002121:Calcium Channel Blockers; D018678:Cholinergic Agents; D020916:Conotoxins; C075400:Cytisus-type anti-H(O) lectins; D018722:Nicotinic Agonists; D037121:Plant Lectins; D021122:Protein Subunits; D011725:Pyridines; D011978:Receptors, Nicotinic; C485184:enhanced green fluorescent protein; C104518:nicotinic receptor alpha6; D049452:Green Fluorescent Proteins; D009538:Nicotine; C082748:epibatidine; D000109:Acetylcholine
keywords: ACh; Acetylcholine; Alpha6; Beta2; Electrophysiology; Nicotinic; Receptors; TM; nAChR; nicotinic acetylcholine receptor; transmembrane segment; α6(⁎); α6-containing