N-GLYCOSYLATION AT THE CONSERVED SITES ENSURES THE EXPRESSION OF PROPERLY FOLDED FUNCTIONAL ACH RECEPTORS

The role of the conserved carbohydrate moiety in the expression of com plete acetylcholine receptor (AChR), alpha(2) beta gamma delta, was re -investigated by expressing additional site-directed mutant subunits, lacking an N-glycosylation site, in Xenopus oocytes. All mutant subuni ts were stably expressed and appeared to associate with other normal s ubunits; however, removal of carbohydrate on the alpha subunit inhibit ed the formation of I-125-alpha-bungarotoxin (alpha-BuTX) binding site s and functional ACh-gated ion channels. I-125-alpha-BuTX binding to A ChRs was also significantly reduced by removal of the conserved carboh ydrate on the gamma or delta subunits. Immunoprecipitation with monocl onal antibodies that recognize the two distinct alpha-BuTX sites on th e AChR indicated that the mutant gamma subunit did not interfere with efficient formation of the alpha-BuTX binding site at the alpha/delta interface, but loss of the carbohydrate did interfere with formation o f the alpha-BuTX binding site at the alpha/mutant gamma interface. A s imilar result was obtained with the mutant delta subunit. Furthermore, the mutant gamma and mutant delta subunits were not incorporated effi ciently into the mature (correct tertiary conformation capable of alph a-BuTX binding) alpha beta delta or alpha beta gamma complexes, respec tively. Since both mutant gamma and mutant delta subunits were capable of assembling with the alpha subunits (immature assembly), these resu lts suggest that the formation of the two alpha-BuTX binding sites req uires correct folding of the alpha gamma and alpha delta complexes, wh ich is aided by the conserved carbohydrate on the gamma and delta subu nits. Electrophysiological experiments demonstrated that functional re ceptors containing mutant subunits were produced, but the functional p roperties of the mutant receptors were differentially altered, dependi ng on the subunit mutated. Together, our results suggest that Ai-glyco sylation of AChR subunits ensures the correct folding of important fun ctional domains and expression of proper functional receptors in the p lasma membrane.