β3 subunits promote expression and nicotine-induced up-regulation of human nicotinic α6*nicotinic acetylcholine receptors expressed in transfected cell lines

Nicotinic acetylcholine receptors (AChRs) containing alpha 6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha 6* AChRs usually contain beta 3 subunits. beta 3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha 6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha 6 beta 2 beta 3 and alpha 6 beta 4 beta 3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha 6 beta 2 or alpha 6 beta 4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a alpha 3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha 6 beta 2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha 6 beta 2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha 6 and beta 3 subunits to aid in the characterization of these AChRs. The alpha 6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta 3 can play important roles in alpha 6* AChR expression, stability, and up-regulation by nicotine.