INHIBITION OF GLUCOSE TRIMMING WITH CASTANOSPERMINE REDUCES CALNEXIN ASSOCIATION AND PROMOTES PROTEASOME DEGRADATION OF THE ALPHA-SUBUNIT OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR

To identify factors involved in the expression of ligand-gated ion cha nnels, we expressed nicotinic acetylcholine receptors in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acety lcholine receptor traverse the membrane four times, contain at least o ne oligosaccharide, and are retained in the endoplasmic reticulum unti l completely assembled into the circular arrangement of subunits of de lta-alpha-gamma-alpha-beta to enclose the ion channel. We previously d emonstrated that calnexin is associated with unassembled subunits of t he receptor, but appears to dissociate when subunits are assembled in various combinations. We used the glucosidase inhibitor castanospermin e to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castan ospermine treatment reduces the association of calnexin with the alpha -subunit of the receptor, and diminishes the intracellular accumulatio n of unassembled receptor subunit protein. However, treatment with cas tanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subun it protein. Co-transfection of cDNAs encoding the alpha- and delta-sub units, which leads to the expression of assembled alpha- and delta- su bunits, also inhibits the loss of cu-subunits expressed in the presenc e of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway.