每个同源α中最外层的脂质暴露α-螺旋(M4),β,δ,和肌肉烟碱乙酰胆碱受体(nAChR)的γ/ε亚基先前已被提出用作脂质传感器。然而,这个传感器的功能机制尚不清楚。探讨成人肌肉nAChR中每个亚基的M4α螺旋如何影响功能,从而探索其在脂质传感中的作用,我们在功能上表征了αM4,βM4,δM4和εM4中每个残基的丙氨酸突变,以及每个亚基M4后区域的丙氨酸和缺失突变。尽管没有发现涉及M4或M4后残基的关键相互作用,我们发现M4-M1/M3界面的大量突变改变了激动剂诱导的反应.此外,发现M4中不同亚基的同源突变对通道功能有不同的影响。还发现沿着一个亚基的M4或在不同亚基的M4同源位置的多个突变的功能效应是加性的。最后,当在非洲爪狼卵母细胞和人胚肾293T细胞中表征时,选择αM4突变显示的细胞特异性表型,可能是因为不同的膜脂环境。总的来说,我们的数据表明M4α螺旋在每个异聚nAChR亚基中的不同功能作用,并预测涉及M4的脂质传感主要通过M4-M1/M3界面的累积相互作用发生,与改变对通道功能至关重要的特定相互作用相反。
The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4-M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4-M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function.