title: Characterizing functional α6β2 nicotinic acetylcholine receptors in vitro: mutant β2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells.
abstract: α6* nicotinic acetylcholine receptors (nAChRs) are highly expressed in mesostriatal and nigrostriatal dopaminergic systems, and participate in motor control, reward, and learning and memory. In vitro functional expression of α6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-α6 and β2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 μM ACh in 26% (5/19) of cells with evenly expressed α6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional α6-eGFPβ2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into β2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased α6-eGFPβ2 nAChR current amplitude. α6-eGFPβ2 nAChRs were also activated by nicotine and by TC-2403. The α6-eGFPβ2 currents were desensitized by 1μM nicotine, blocked by α-conotoxin MII, partially inhibited by dihydro-β-erythroidine, and potentiated by extracellular Ca(2+). Single-channel recordings showed that α6-eGFPβ2 nAChRs had similar single-channel conductance to, but longer open time than, α4-eGFPβ2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of α6* nAChRs for both pharmacological study and drug screening.
authors: California Institute of Technology, 156-29 Caltech, Pasadena, CA 91125, USA.
authors: Cheng
authors: C
authors: Xiao
authors: Rahul
authors: R
authors: Srinivasan
authors: Ryan M
authors: RM
authors: Drenan
authors: Elisha D W
authors: ED
authors: Mackey
authors: J Michael
authors: JM
authors: McIntosh
authors: Henry A
authors: HA
authors: Lester
references: J Pharmacol Exp Ther. 2009 Nov;331(2):547-54
references: 19644040
references: J Pharmacol Exp Ther. 2005 Feb;312(2):619-26
references: 15356217
references: J Neurosci. 2003 Apr 15;23(8):3176-85
references: 12716925
references: Mol Pharmacol. 2007 Jun;71(6):1563-71
references: 17341654
references: Neuron. 2008 Oct 9;60(1):4-7
references: 18940582
references: Mol Pharmacol. 2008 Sep;74(3):844-53
references: 18583454
references: Circ Res. 2006 Feb 3;98(2):245-53
references: 16357306
references: Brain Res Brain Res Rev. 2005 Jun;48(3):420-37
references: 15914250
references: Mol Pharmacol. 2008 Jan;73(1):27-41
references: 17932221
references: EMBO Rep. 2007 Mar;8(3):258-64
references: 17255961
references: Mol Pharmacol. 2009 May;75(5):1137-48
references: 19237585
references: J Pharmacol Exp Ther. 2006 Feb;316(2):481-9
references: 16210393
references: Curr Protoc Cytom. 2009 Oct;Chapter 12:Unit12.18
references: 19816922
references: Brain Res Rev. 2007 Aug;55(1):134-43
references: 17383007
references: Nature. 1997 Nov 27;390(6658):401-4
references: 9389479
references: Mol Pharmacol. 2006 Aug;70(2):755-68
references: 16720757
references: J Biol Chem. 2004 Sep 3;279(36):38007-15
references: 15247303
references: J Neurosci. 2008 Nov 19;28(47):12318-27
references: 19020025
references: Mol Pharmacol. 2002 Jan;61(1):127-35
references: 11752213
references: J Neurosci. 2010 Jul 21;30(29):9877-89
references: 20660270
references: Mol Pharmacol. 2004 Jun;65(6):1526-35
references: 15155845
references: J Neurosci. 2009 Oct 7;29(40):12428-39
references: 19812319
references: Br J Pharmacol. 2010 Aug;160(8):1906-15
references: 20649589
references: Neuron. 2008 Oct 9;60(1):123-36
references: 18940593
references: Neuron. 2002 Mar 14;33(6):905-19
references: 11906697
references: J Neurophysiol. 2000 Mar;83(3):1329-37
references: 10712460
references: J Neurosci. 2001 Mar 15;21(6):1819-29
references: 11245666
references: J Pharmacol Exp Ther. 1997 Jan;280(1):346-56
references: 8996215
references: Mol Pharmacol. 2003 Feb;63(2):332-41
references: 12527804
references: Science. 2004 Nov 5;306(5698):1029-32
references: 15528443
references: BMC Neurosci. 2009 Nov 30;10:141
references: 19948025
references: Mol Pharmacol. 2006 Oct;70(4):1358-68
references: 16835356
references: Mol Pharmacol. 2011 Jan;79(1):126-40
references: 20923852
references: Biochem Pharmacol. 2007 Oct 15;74(8):1235-46
references: 17825262
references: Mol Pharmacol. 2005 Aug;68(2):487-501
references: 15901849
references: Biophys J. 2009 Apr 8;96(7):2949-60
references: 19348776
references: J Biol Chem. 2008 Mar 7;283(10):6022-32
references: 18174175
references: Neuropharmacology. 2000 Oct;39(13):2570-90
references: 11044728
references: Mol Pharmacol. 1995 Oct;48(4):774-82
references: 7476906
references: Neuropharmacology. 2010 Jun;58(7):1054-66
references: 20114055
references: J Physiol Paris. 1992;86(1-3):67-76
references: 1285313
references: Mol Pharmacol. 1997 Feb;51(2):320-7
references: 9203638
references: EMBO J. 2008 Nov 5;27(21):2918-28
references: 18843296
references: J Biol Chem. 2006 Mar 24;281(12):8062-71
references: 16407231
references: J Neurosci. 1996 Dec 15;16(24):7880-91
references: 8987816
references: J Gen Physiol. 2011 Jan;137(1):59-79
references: 21187334
references: Neuropsychopharmacology. 2010 Feb;35(3):665-73
references: 19890263
references: Prog Brain Res. 1990;83:197-214
references: 2168056