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

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Abstract
Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing α6 subunits (α6* nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human α6β2, α6β4, α6β2β3, α6β4β3, or α6β4β3α5 nAChR. Cells expressing subunits needed to form α6β4β3α5 nAChR exhibited saturable [3H]epibatidine binding (Kd = 95.9 ± 8.3 pM and Bmax = 84.5 ± 1.6 fmol/mg of protein). The rank order of binding competition potency (Ki) for prototypical nicotinic compounds was α-conotoxin MII (6 nM) > nicotine (156 nM) ∼ methyllycaconitine (200 nM) > α-bungarotoxin (>10 μM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that β3 and α5 subunits are likely partners of the α6 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 α6 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 α5 subunit makes a critical contribution to α6β4β3α5 nAChR assembly into a ligand-binding form with native α6*-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.

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