Gehle V M, Walcott E C, Nishizaki T, Sumikawa K
Department of Psychobiology, University of California at Irvine, 92697-4550, USA.
Brain Res Mol Brain Res. 1997 May;45(2):219-29. doi: 10.1016/s0169-328x(96)00256-2.
The role of the conserved carbohydrate moiety in the expression of complete acetylcholine receptor (AChR), alpha2 beta gamma delta was re-investigated by expressing additional site-directed mutant subunits, lacking an N-glycosylation site, in Xenopus oocytes. All mutant subunits were stably expressed and appeared to associate with other normal subunits; however, removal of carbohydrate on the alpha subunit inhibited the formation of 125I-alpha-bungarotoxin (alpha-BuTX) binding sites and functional ACh-gated ion channels. 125I-alpha-BuTX binding to AChRs was also significantly reduced by removal of the conserved carbohydrate on the gamma or delta subunits. Immunoprecipitation with monoclonal antibodies that recognize the two distinct alpha-BuTX sites on the 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 of the alpha-BuTX binding site at the alpha/mutant gamma interface. A similar result was obtained with the mutant delta subunit. Furthermore, the mutant gamma and mutant delta subunits were not incorporated efficiently into the mature (correct tertiary conformation capable of alpha-BuTX binding) alpha beta delta or alpha beta gamma complexes, respectively. Since both mutant gamma and mutant delta subunits were capable of assembling with the alpha subunits (immature assembly), these results suggest that the formation of the two alpha-BuTX binding sites requires correct folding of the alpha gamma and alpha delta complexes, which is aided by the conserved carbohydrate on the gamma and delta subunits. Electrophysiological experiments demonstrated that functional receptors containing mutant subunits were produced, but the functional properties of the mutant receptors were differentially altered, depending on the subunit mutated. Together, our results suggest that N-glycosylation of AChR subunits ensures the correct folding of important functional domains and expression of proper functional receptors in the plasma membrane.
通过在非洲爪蟾卵母细胞中表达额外的缺乏N-糖基化位点的定点突变亚基，对保守碳水化合物部分在完整乙酰胆碱受体（AChR）α2βγδ表达中的作用进行了重新研究。所有突变亚基均稳定表达，且似乎能与其他正常亚基结合；然而，α亚基上碳水化合物的去除抑制了125I-α-银环蛇毒素（α-BuTX）结合位点和功能性ACh门控离子通道的形成。γ或δ亚基上保守碳水化合物的去除也显著降低了125I-α-BuTX与AChR的结合。用识别AChR上两个不同α-BuTX位点的单克隆抗体进行免疫沉淀表明，突变γ亚基不干扰α/δ界面处α-BuTX结合位点的有效形成，但碳水化合物的缺失确实干扰了α/突变γ界面处α-BuTX结合位点的形成。突变δ亚基也得到了类似结果。此外，突变γ亚基和突变δ亚基分别没有有效地整合到成熟的（能够结合α-BuTX的正确三级构象）αβδ或αβγ复合物中。由于突变γ亚基和突变δ亚基都能够与α亚基组装（不成熟组装），这些结果表明，两个α-BuTX结合位点的形成需要αγ和αδ复合物的正确折叠，而γ和δ亚基上的保守碳水化合物有助于这种折叠。电生理实验表明，产生了含有突变亚基的功能性受体，但突变受体的功能特性根据突变的亚基不同而有差异地改变。总之，我们的结果表明，AChR亚基的N-糖基化确保了重要功能域的正确折叠以及质膜中正常功能性受体的表达。
