Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.
Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.
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DOI：
10.1016/s0021-9258(18)42693-2
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发表时间：
1992-03
期刊：
影响因子：
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通讯作者：
Katumi Sumikawa;Vaughn M. Gehle
中科院分区：
文献类型：
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作者：
Katumi Sumikawa;Vaughn M. Gehle
烟碱乙酰胆碱受体（AChR）的每个亚基含有两个保守的半胱氨酸残基，已知它们在n端胞外区域形成二硫键。通过在爪蟾卵母细胞中表达加州鱼雷AChR的位点定向突变体α和β亚基以及其他正常亚基，研究了这种保留的结构特征在AChR生物发生中的作用。α亚基的128或142位或β亚基的半胱氨酸突变不会阻止亚基的组装。α亚基和β亚基的所有Cys128和Cys142突变体都能与共表达的其他正常亚基结合，尽管突变体α亚基与δ亚基的结合效率降低。突变体AChR复合物的功能研究表明，α亚基的突变使整个卵母细胞中可检测到的125i - α -bungarotoxin （α - butx）结合消失，而β亚基的突变导致125i - α - butx的总结合减少，表面125i - α - butx无法检测到。此外，当所有突变亚基在卵母细胞中与其他正常亚基共表达时，产生小的乙酰胆碱激活的膜电流，这表明只有少量功能突变的achr掺入质膜。α - butx不能阻断突变体α亚基而非突变体β亚基形成的功能性乙酰胆碱门控离子通道。因此，AChR α或β亚基的Cys128和Cys142之间的二硫键不需要与乙酰胆碱结合。然而，α亚基上的二硫键是形成α - butx结合位点所必需的。这些结果还表明，破坏保守的二硫环结构所造成的最显著的影响是大多数组装的AChR复合物在细胞内保留。
Each subunit of the nicotinic acetylcholine receptor (AChR) contains two conserved cysteine residues, which are known to form a disulfide bond, in the N-terminal extracellular domain. The role of this retained structural feature in the biogenesis of the AChR was studied by expressing site-directed mutant alpha and beta subunits together with other normal subunits from Torpedo californica AChR in Xenopus oocytes. Mutation of the cysteines at position 128 or 142 in the alpha subunit, or in the beta subunit, did not prevent subunit assembly. All Cys128 and Cys142 mutants of the alpha and beta subunits were able to associate with coexpressed other normal subunits, although associational efficiency of the mutant alpha subunits with the delta subunit was reduced. Functional studies of the mutant AChR complexes showed that the mutations in the alpha subunit abolished detectable 125I-alpha-bungarotoxin (alpha-BuTX) binding in whole oocytes, whereas the mutations in the beta subunit resulted in decreased total binding of 125I-alpha-BuTX and no detectable surface 125I-alpha-BuTX binding. Additionally, all mutant subunits, when co-expressed with the other normal subunits in oocytes, produced small acetylcholine-activated membrane currents, suggesting incorporation of only small numbers of functional mutant AChRs into the plasma membrane. The functional acetylcholine-gated ion channel formed with mutant alpha subunits, but not mutant beta subunits, could not be blocked by alpha-BuTX. Thus, a disulfide bond between Cys128 and Cys142 of the AChR alpha or beta subunits is not needed for acetylcholine-binding. However, this disulfide bond on the alpha subunit is necessary for formation of the alpha-BuTX-binding site. These results also suggest that the most significant effect caused by disrupting the conserved disulfide loop structure is intracellular retention of most of the assembled AChR complexes.