Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics
Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics
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DOI：
10.1124/mol.54.1.146
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发表时间：
1998-07-01
影响因子：
3.6
通讯作者：
Barrantes, FJ
中科院分区：
文献类型：
--
作者：
Bouzat, C;Roccamo, AM;Barrantes, FJ
烟碱乙酰胆碱受体（AChR）的跨膜部分中与脂质双层接触的最牢固的候选者是第四段，M4。为了探索小鼠AChR的α M4氨基酸残基对通道门控的贡献，我们将定点突变与单通道记录相结合。发现α M4中的两个残基Cys 418和Thr 422在被丙氨酸取代时显著影响门控动力学。含有α C418 A和α T422 A亚基的AChR形成通道，其特征在于平均开放时间分别减少3倍和5倍，表明由于突变导致关闭率增加。计算出的通道关闭过程中的能量势垒的变化显示不平等和耦合的贡献，这两个位置通道门控。杂交野生型α/α T422 A AChR的单通道记录表明，关闭率取决于α亚基突变的数量。每一个苏氨酸取代丙氨酸改变关闭过程的能量障碍，通过类似的0.5千卡/摩尔。高浓度激动剂激活的通道的记录表明，这些突变也损害通道开放。Cys 418和Thr 422都被假定与脂质环境接触，并且在物种和亚基之间高度保守。我们的研究结果支持脂质暴露的残基在AChR通道门控机制的α M4参与。
The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of alpha M4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in alpha M4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing alpha C418A and alpha T422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type alpha/alpha T422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the closing process by similar to 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in alpha M4 in AChR channel gating mechanism.