Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating.
Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating.
复制标题
烟碱受体 M1 跨膜结构域中残基对通道门控的机制贡献。
DOI：
10.1080/09687680310001607341
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Bouzat,Cecilia
中科院分区：
文献类型：
--
作者：
Spitzmaul,Guillermo;Corradi,Jeremias;Bouzat,Cecilia
烟碱受体（nicotinic receptor，AChR）是一个由α2βεδ亚基组成的五聚体。每个亚基含有四个跨膜结构域（M1-M4）。M1结构域的15′位在α亚基中是苯丙氨酸，而在非α亚基中是异亮氨酸。鉴于这种特殊的保护模式，我们研究了它的贡献，肌肉AChR激活相结合的诱变单通道动力学分析。与野生型AChR相比，含有突变α亚基（αF15′I）以及含有非α亚基（βI15′F、δI15′F和εI15′F）的AChR显示出由于关闭速率较慢而导致的双配体开放通道的寿命延长。亚基之间的动力学变化是不相等的，β亚基是产生最显著的开放状态稳定化的亚基。动力学分析表明，在低效激动剂胆碱作用下，βI15′ F-AChR通道的关闭速率降低10倍，开放速率增加2.5倍，门控平衡常数增加28倍，无激动剂时开放速率显著增加。βI15′突变表明其参与门控的结构基础是复杂的。速率-平衡线性自由能关系表明，β15′位在门控过渡态处处于一个近似70%封闭态的环境.总体结果确定位置15 '作为通道门控的亚基选择性决定因素，并增加了新的实验证据，支持M1结构域参与通道门控装置的操作。
The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1–M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.