Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker
Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker
复制标题
DOI：
10.1093/brain/awh364
复制
发表时间：
2005-02-01
期刊：
影响因子：
14.5
通讯作者：
Engel, AG
中科院分区：
文献类型：
--
作者：
Shen, XM;Ohno, K;Engel, AG
我们追踪两例先天性肌无力综合征患者的原因，发现其与肌肉乙酰胆碱受体（AChR）α亚基突变有关。这两名患者都在连接第三（M3）和第四（M4）跨膜结构域的胞质环的C-末端的α亚基（ε N436 del）中缺失天冬酰胺残基。在ε亚基的第二等位基因中存在无效突变表明ε N346 del决定了表型。终板研究显示epsilonN 346 del-AChR的表达明显减少，胎儿γ-AChR代偿性蓄积。HEK细胞中的表达研究揭示了ε N436 del-AChR的表达降低和异常短暂的通道开放。因此，神经肌肉传递受到AChR缺陷、ε N346 del-AChR的快通道动力学和γ-AChR的不完全表型拯救的损害。单通道动力学分析表明，epsilonN 436 del缩短通道开放，通过降低稳定性的受体：通道关闭和ACh解离的速率增加，通道开放的速率降低。除了缩短M3-M4环之外，ε N436 del还移动了与M4相邻的带负电荷的天冬氨酸残基; ε N436 del的作用显示是由于M3-M4环的缩短而不是由于负电荷与M4的并置。为了确定ε N346缺失的结果是否是亚基特异性的，我们删除了β、δ和α亚基中与ε N436对齐的残基。每个缺失突变体减少AChR表达，但β和δ突变体缩短通道开放时间，α突变体显著缩短通道开放时间。动力学分析表明，α突变体增加了双配体受体的稳定性：通道关闭和ACh解离的速率降低，通道开放的速率增加。整体研究揭示了亚基不对称的贡献M3-M4循环在优化AChR激活通过变构连接的通道和激动剂结合位点。
We trace the cause of congenital myasthenic syndromes in two patients to mutations in the epsilon subunit of the muscle acetylcholine receptor (AChR). Both patients harbour deletion of an asparagine residue in the epsilon subunit (epsilonN436del) at the C-terminus of the cytoplasmic loop linking the third (M3) and fourth (M4) transmembrane domains. The presence of a null mutation in the second allele of the epsilon subunit shows that epsilonN346del determines the phenotype. Endplate studies show markedly reduced expression of the epsilonN346del-AChR and compensatory accumulation of fetal gamma-AChR. Expression studies in HEK cells reveal decreased expression of epsilonN436del-AChR and abnormally brief channel openings. Thus, neuromuscular transmission is compromised by AChR deficiency, fast channel kinetics of the epsilonN346del-AChR and incomplete phenotypic rescue by gamma-AChR. Single-channel kinetic analysis shows that the epsilonN436del shortens channel openings by reducing stability of the diliganded receptor: rates of channel closing and of ACh dissociation are increased and the rate of channel opening is decreased. In addition to shortening the M3-M4 loop, epsilonN436del shifts a negatively charged aspartic acid residue adjacent to M4; the effects of epsilonN436del are shown to result from shortening of the M3-M4 loop and not from juxtaposition of a negative charge to M4. To determine whether the consequences of epsilonN346del are subunit-specific, we deleted residues that align with epsilonN436 in beta, delta and alpha subunits. Each deletion mutant reduces AChR expression, but whereas the beta and delta mutants curtail channel open duration, the alpha mutant strikingly prolongs open duration. Kinetic analysis reveals that the alpha mutant increases the stability of the diliganded receptor: rates of channel closing and of ACh dissociation are decreased and the rate of channel opening is increased. The overall studies reveal subunit asymmetry in the contributions of the M3-M4 loops in optimizing AChR activation through allosteric links to the channel and the agonist binding site.