Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor α subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type α subunit acquires high affinity α bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of a subunit as assayed by high affinity toxin binding. The unassembled wild-type α subunit expressed in fibroblasts is normally degraded with a t_1/2 of 2 h; upon assembly with the α subunit, the degradation rate slows significantly (t_1/2 > 13 h). All mutated α subunits retained the capacity to assemble with a δ subunit coexpressed in fibroblasts; however, mutated α subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t_1/2 = 20 min to 2 h) regardless of whether or not they assembled with the δ subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin-treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR-expressing cells.