Heterologous Expression of Human α6β4β3α5 Nicotinic Acetylcholine Receptors: Binding Properties Consistent with Their Natural Expression Require Quaternary Subunit Assembly Including the α5 Subunit

Abstract

Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing ␣6 subunits (␣6* nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human ␣6␤2, ␣6␤4, ␣6␤2␤3, ␣6␤4␤3, or ␣6␤4␤3␣5 nAChR. Cells expressing subunits needed to form ␣6␤4␤3␣5 nAChR exhibited saturable [ 3 H]epibatidine binding (K d ϭ 95.9 Ϯ 8.3 pM and B max ϭ 84.5 Ϯ 1.6 fmol/mg of protein). The rank order of binding competition potency (K i ) for prototypical nicotinic compounds was ␣-conotoxin MII (6 nM) Ͼ nicotine (156 nM) ϳ methyllycaconitine (200 nM) Ͼ ␣-bungarotoxin (Ͼ10 M), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that ␤3 and ␣5 subunits are likely partners of the ␣6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that ␣6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human ␣5 subunit makes a critical contribution to ␣6␤4␤3␣5 nAChR assembly into a ligand-binding form with native ␣6*-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.Distinct nicotinic acetylcholine receptor (nAChR) subtypes expressed in mesostriatal dopaminergic neurons are involved in modulation of striatal dopamine (DA) release (Wonnacott, 1997;Champtiaux et al., 2002;Luetje, 2004); nAChR containing ␣4 and ␤2 subunits (␣4␤2* nAChR) or containing ␣6 subunits (␣6* nAChR) participate directly, and ␣7* nAChR participate indirectly. Whereas the biochemistry of ␣4␤2* and ␣7* nAChR is well characterized, the subunit composition and functional properties of ␣6* nAChR remain unclear. Suggested roles for ␣6* nAChR in modulation of DA transmission imply their potential importance in locomotion, reward, schizophrenia, and Parkinson's disease (le Novère et al

Pharmacological characterisation of α6β4⁎ nicotinic acetylcholine receptors assembled from three chimeric α6/α3 subunits in tsA201 cells

“…When it comes to binding properties, the rank order of binding affinities displayed by the seven agonists at WT α3β4 resemble those previously reported for this receptor (Jensen et al, 2003;Rollema et al, 2007;Zwart et al, 2008), and the K i values obtained for (7 )-epibatidine, acetylcholine, carbachol, (S)-nicotine and ( À )-cytisine at the C1β4, C6 F223L β4 and C16 F223L β4 receptors are comparable to those reported for human α6β3β4α5 and chick α6β4 receptors (Barabino et al, 2001;Grinevich et al, 2005). The rank order of agonist potencies at WT α3β4 in the FLIPR™ Membrane Potential Blue assay concords with previous studies of the receptor in this or other fluorescence-based assays (Fitch et al, 2003;Jensen et al, 2003;Stauderman et al, 1998).…”

Nicotinic receptor stimulation protects nigral dopaminergic neurons in rotenone‐induced Parkinson's disease models

“…The concentration of each antagonist was the maximal dosage that did not show cytotoxicity or interference among nAChRs. Ki values of aBuTx were 2.16 (1.56-3.01) nM for a7 nAChR and >10,000 nM for a4b2nAChR, and those of DHbE were 7700 (4510-13,100) nM for a7 nAChR and 24.6 (16.9-35.8) nM for a4b2 nAChR (Grinevich et al, 2005). To confirm PI3K inhibition, LY294002 was administrated 10 min before the additional treatment of rotenone and nicotine.…”

Incorporation of the β3 Subunit Has a Dominant-Negative Effect on the Function of Recombinant Central-Type Neuronal Nicotinic Receptors

“…Robust responses were observed only when the gain-of-function mutant ␤3 VS was coexpressed. A further complication is that efficient surface expression of ␣6␤4 ligand binding sites may require both the ␤3 and the ␣5 subunit (Grinevich et al, 2005).…”