Received for publication February 29, 2008.
Revised March 26, 2008.
Accepted for publication March 31, 2008.

Roles of Accessory Subunits in 42* Nicotinic Receptors

Abstract

Accessory subunits in heteromeric nicotinic receptors (AChRs) do not take part in forming ACh binding sites. 5 and 3 subunits can function only as accessory subunits. We show that both 5 and 3 efficiently assemble in human 42* AChRs expressed in permanently transfected HEK cell lines. Only (42)₂5, not (42)₂3 AChRs, have been detected in brain. The 425 line expressed 40% more AChRs than the parent 42 line, and was equally sensitive to upregulation by nicotine. The 423 line expressed 25 fold more AChRs than the parental line, and could not be further upregulated by nicotine. Relative sensitivity to activation by ACh depends on the accessory subunit, with 2 conferring the greatest sensitivity, 5 less, and 3 and 4 much less. Accessory subunits form binding sites for positive allosteric modulators, as illustrated by the observation that 5 conferred high sensitivity to galanthamine. In the presence of 5 or 3, stable, partially degraded, dead end intermediates accumulated within the cells. These may have of the form 5425. The efficiency with which 5 and 3 assemble with 4 and 2, and the necessity of avoiding formation of potentially toxic intermediates, may explain why 5 and 3 appear to be transcribed at low levels in brain. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) can be caused by the 4 mutation S247F. This mutant did not produce functional AChRs unless cells were cotransfected with 5, 3, or 6 to replace 4 as accessory subunit.

Key words: Nicotinic cholinergic, Func. analysis receptor/ion channel mutants, Receptor binding studies

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