Roles of accessory subunits in α4β2*nicotinic receptors

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