ASSEMBLY OF TORPEDO ACETYLCHOLINE-RECEPTORS IN XENOPUS OOCYTES

To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo alpha-subunits were expressed in Xenopus oocytes alone or in combination with beta, gamma, or delta-subunits. The maturation of the conformation of the main immunogenic region (MIR) on alpha-subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on alpha-subunits was measured by binding of alpha-bungarotoxin (alpha-Bgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition alpha-2-beta-gamma-delta. Torpedo alpha-subunits expressed alone resulted in an amorphous range of complexes with little affinity for alpha-Bgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in alpha-subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured alpha-subunits. Coexpression of alpha with beta-subunits also resulted in an amorphous range of complexes. However, coexpression of alpha-subunits with gamma or delta-subunits resulted in the efficient formation of 6.5S-alpha-gamma or alpha-delta-complexes with high affinity for mAbs to the MIR, alpha-Bgt, and small cholinergic ligands. These alpha-gamma and alpha-delta-subunit pairs may represent normal assembly intermediates in which Torpedo-alpha is stabilized and matured in conformation. Coexpression of alpha, gamma, and delta-efficiently formed 8.8S complexes, whereas complexes containing alpha-beta and gamma or alpha-beta and delta-subunits are formed less efficiently. Assembly of beta-subunits with complexes containing alpha-gamma and delta-subunits may normally be a rate-limiting step in assembly of AChRs.