Received for publication June 1, 2006.
Revised July 3, 2006.
Accepted for publication July 11, 2006.

3 Subunits Promote Expression and Nicotine- Induced Upregulation of Human Nicotinic 6* AChRs Expressed in Transfected Cell Lines

Abstract

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. 6* AChRs usually contain 3 subunits. 3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming ACh binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human 6 AChRs subtypes were permanently transfected into human tsA201 HEK cell lines. 623 and 64& [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]62 or 64 cell lines. The increased sensitivity to nicotine-induced upregulation was not due to a 3 induced increase in affinity for nicotine, but probably due to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature 62 AChRs, but many of these are on the cell surface. This contrasts with Xenopus oocytes which express a large amount of incorrectly assembled 62 subunits which bind cholinergic ligands but form large amorphous intracellular aggregates. mAbs were made to the 6 and 3 subunits to aid in the characterization of these AChRs. The 6 mAbs bind to epitopes C- terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit 3 can play important roles in 6* AChR expression, stability, and upregulation by nicotine.

Key words: Nicotinic cholinergic, Receptor binding studies

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