Abstract
The muscle-type nicotinic acetylcholine receptor (AChR)2 is a pentameric membrane ion channel assembled in the endoplasmic reticulum from four homologous subunits by mechanisms that are insufficiently understood. Nascent AChR subunits were recently found to form complexes with the endoplasmic reticulum-resident molecular chaperone calnexin. To determine the contribution of this interaction to AChR assembly and surface expression, we have now used transient transfection of mouse AChR subunits and calnexin into non-muscle cells. Co-transfection of calnexin along with AChR subunits into COS and HEK 293 cells was found to enhance AChR subunit folding and assembly, and to decrease degradation rates of newly synthesized AChR alpha-subunits, resulting in elevated surface expression of assembled AChR. Moreover, inhibition of the interaction between endogenous calnexin and AChR by castanospermine resulted in decreased AChR subunit folding, assembly, and surface expression in muscle and HEK 293 cells. Together, these findings provide evidence that calnexin directly contributes to AChR biogenesis by promoting subunit folding and assembly.
Highlights
Many of the transmembrane signaling molecules present in cell surface membranes are oligomeric proteins, and assembly of these proteins from constituent subunits is required for their functional expression
In the present study we have examined the contribution of calnexin to acetylcholine receptor (AChR) biogenesis in two ways: first, we took advantage of the inefficient folding and assembly of recombinant AChR subunits in transfected cells to determine if co-transfection of exogenous calnexin into these cells can increase AChR expression
The unassembled folded AChR ␣-subunit was quantified as the component of 125I-Bgt binding that is preserved in the presence of carbamylcholine, after subtraction of nonspecific binding estimated by the quantification of 125I-Bgt bound to nontransfected cultures
Summary
Assembly of AChR subunits in the ER has been lacking. In the present study we have examined the contribution of calnexin to AChR biogenesis in two ways: first, we took advantage of the inefficient folding and assembly of recombinant AChR subunits in transfected cells to determine if co-transfection of exogenous calnexin into these cells can increase AChR expression. The ability of the glucose trimming inhibitor castanospermine (CAS) to block the interaction of endogenous calnexin with its substrates [28] was utilized to examine the effect of this block on AChR biogenesis
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