OBJECTIVE Direct inhibition of acetylcholine receptor (AChR) function by autoantibodies (Abs) is considered a rare pathogenic mechanism in myasthenia gravis (MG), but is usually studied on AChRs expressed in cell lines, rather than tightly clustered by the intracellular scaffolding protein, rapsyn, as at the intact neuromuscular junction. We hypothesised that clustered AChRs would provide a better target for investigating the functional effects of AChR-Abs. METHODS Acetylcholine-induced currents were measured using whole-cell patch clamping and a fast perfusion system to assess fast (<2 min) functional effects of the serum samples. The sensitivity, specificity and rapidity of the system were first demonstrated by applying maternal AChR-Ab positive plasmas known to inhibit fetal AChR function in TE671 cells. Eleven previously untested AChR-Ab positive MG sera, 10 AChR-Ab negative MG sera and 5 healthy control sera were then applied to unclustered and rapsyn-clustered human adult AChRs in CN21 cells. RESULTS The maternal AChR-Ab positive plasmas reduced fetal AChR currents, but not adult AChR currents, by >80% within 100 s. Only 2/11 AChR-Ab positive sera inhibited AChR currents in unclustered AChRs, but 6/11 AChR-Ab positive sera compared with none of the 10 AChR-Ab negative sera (p=0.0020) inhibited rapsyn-clustered AChR currents, and current inhibition by the AChR-Ab positive sera was greater when the AChRs were clustered (p=0.0385). None of the sera had detectable effects on desensitisation or recovery from desensitisation. CONCLUSION These results show that antibodies can inhibit AChR function rapidly and demonstrate the importance of clustering in exploring pathogenic disease mechanisms of MG Abs.

中文翻译：

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重症肌无力 AChR 抗体抑制 rapsyn 簇 AChR 的功能。

目的 自身抗体 (Abs) 对乙酰胆碱受体 (AChR) 功能的直接抑制被认为是重症肌无力 (MG) 的一种罕见致病机制，但通常对细胞系中表达的 AChRs 进行研究，而不是由细胞内支架蛋白 rapsyn 紧密聚集，如在完整的神经肌肉接头处。我们假设成簇的 AChR 将为研究 AChR-Ab 的功能影响提供更好的目标。方法使用全细胞膜片钳和快速灌注系统测量乙酰胆碱诱导的电流，以评估血清样品的快速（<2 分钟）功能影响。该系统的敏感性、特异性和快速性首先通过应用已知可抑制 TE671 细胞中胎儿 AChR 功能的母体 AChR-Ab 阳性血浆得到证明。然后将 11 份先前未经测试的 AChR-Ab 阳性 MG 血清、10 份 AChR-Ab 阴性 MG 血清和 5 份健康对照血清应用于 CN21 细胞中的非簇状和 rapsyn 簇状成人 AChR。结果 母体 AChR-Ab 阳性血浆在 100 秒内降低了胎儿 AChR 电流，但不降低成人 AChR 电流 > 80%。只有 2/11 AChR-Ab 阳性血清抑制非聚集 AChR 中的 AChR 电流，但 6/11 AChR-Ab 阳性血清与 10 个 AChR-Ab 阴性血清中没有一个相比 (p = 0.0020) 抑制 rapsyn 聚集 AChR 电流和电流当 AChR 成簇时，AChR-Ab 阳性血清的抑制作用更大 (p=0.0385)。没有一种血清对脱敏或脱敏恢复具有可检测的影响。