A CONSERVED DISULFIDE LOOP FACILITATES CONFORMATIONAL MATURATION IN THE SUBUNITS OF THE ACETYLCHOLINE-RECEPTOR

To examine the structural determinants for the assembly of ligand-gate d receptors, we constructed mutant alpha, beta, gamma and delta subuni ts of the Torpedo acetylcholine receptor (AChR), lacking one of the co nserved cysteine residues which forms a 13-amino acid disulfide loop i n the amino terminal domain of each subunit. Mutant subunits were co-e xpressed with complementary wild-type subunits in Xenopus oocytes. Usi ng subunit-specific antisera and monoclonal antibodies that recognize the two distinct alpha-bungarotoxin (alpha-BuTX) sites on the AChR, we were able to distinguish immature subunit associations from conformat ionally mature AChR complexes. Removal of the disulfide loop on the al pha subunit completely destroyed the formation of the two toxin-bindin g sites, while removal of the structure on the beta subunit had little effect. While mutant gamma and delta subunits were capable of forming associations (immature assembly) with other subunits, the formation o f alpha-BTX sites between alpha and mutant gamma or mutant delta subun its was diminished. Interestingly, assembly of alpha beta gamma subuni ts remained efficient in the presence of mutant delta subunits, wherea s assembly of alpha beta gamma subunits was inefficient in the presenc e of mutant gamma subunits. Thus, these results indicate that the form ation of the disulfide loop facilitates the conformational maturation of alpha gamma and alpha beta gamma complexes, which may be conditiona l for correct subunit coupling in assembling receptors. Furthermore, i t seems likely that the correct coupling between the alpha and gamma s ubunits is the most important step in subunit assembly.