Assembly of Torpedo acetylcholine receptors in Xenopus oocytes

Abstract

To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo a subunits were expressed in Xenopus oocytes alone or in combination with β, γ, or δ subunits. The maturation of the conformation of the main immunogenic region (MIR) on α subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on α subunits was measured by binding of α-bungarotoxin (aBgt) 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 α2βγδ. Torpedo α subunits expressed alone resulted in an amorphous range of complexes with little affinity for αBgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in α subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured α subunits. Coexpression of α with β subunits also resulted in an amorphous range of complexes. However, coexpression of α subunits with γ or δ subunits resulted in the efficient formation of 6.5S αγ or αδ complexes with high affinity for mAbs to the MIR, αBgt, and small cholinergic ligands. These αγ and αδ subunit pairs may represent normal assembly intermediates in which Torpedo α is stabilized and matured in conformation. Coexpression of α, γ, and δ efficiently formed 8.8S complexes, whereas complexes containing αβ and γ or αβ and δsubunits are formed less efficiently. Assembly of β subunits with complexes containing αγ and δsubunits may normally be a ratelimiting step in assembly of AChRs.