Asymmetric and Independent Contribution of the Second Transmembrane Segment 12′ Residues to Diliganded Gating of Acetylcholine Receptor Channels
Asymmetric and Independent Contribution of the Second Transmembrane Segment 12′ Residues to Diliganded Gating of Acetylcholine Receptor Channels
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第二跨膜片段 12 残基对乙酰胆碱受体通道双配体门控的不对称和独立贡献
DOI：
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
A. Auerbach
中科院分区：
文献类型：
--
作者：
C. Grosman;A. Auerbach
突变研究表明，第二跨膜段（M2）在乙酰胆碱受体配体门控过程中起着关键作用。然而，门控和M2结构域的结构之间的关系的充分描述，已受到阻碍的事实，即许多M2突变增加开放速率常数的水平，在乙酰胆碱的存在下，是不可分辨的快。在这里，我们表明，使用饱和浓度的胆碱，一种低效的激动剂，是一个方便的工具来规避这个问题。在20 mM胆碱的存在下：（a）单通道电流出现在簇中;（B）胆碱本身的快速阻断使单通道电导降低约50%，但过量的开放通道噪声仅是中等的;（c）门控动力学通过单步C ParticipO模型拟合得最好;和（d）打开和关闭速率常数在良好可分辨的范围内。将该方法应用于一系列重组成年小鼠肌肉M2 12 '突变体，结果表明：（a）M2 12'的五个同源位置对diliganded gating做出独立且不对称的贡献，其中δ亚基对突变最敏感;（B）δ12′突变以与过渡态对突变的敏感性一致的方式增加双配位门控平衡常数（3）δ12′氨基酸残基体积、疏水性或α-螺旋倾向与相应突变体的门控平衡常数之间的关系并不简单;然而，（d）突变体系列的速率和平衡常数是线性相关的（在对数-对数图上），这表明突变时的构象重排大多是局部的，并且过渡态沿着门控反应坐标的位置不受这些突变的影响。
Mutagenesis studies have suggested that the second transmembrane segment (M2) plays a critical role during acetylcholine receptor liganded gating. An adequate description of the relationship between gating and structure of the M2 domain, however, has been hampered by the fact that many M2 mutations increase the opening rate constant to levels that, in the presence of acetylcholine, are unresolvably fast. Here, we show that the use of saturating concentrations of choline, a low-efficacy agonist, is a convenient tool to circumvent this problem. In the presence of 20 mM choline: (a) single-channel currents occur in clusters; (b) fast blockade by choline itself reduces the single-channel conductance by ∼50%, yet the excess open-channel noise is only moderate; (c) the kinetics of gating are fitted best by a single-step, C ↔ O model; and (d) opening and closing rate constants are within a well resolvable range. Application of this method to a series of recombinant adult mouse muscle M2 12′ mutants revealed that: (a) the five homologous M2 12′ positions make independent and asymmetric contributions to diliganded gating, the δ subunit being the most sensitive to mutation; (b) mutations at δ12′ increase the diliganded gating equilibrium constant in a manner that is consistent with the sensitivity of the transition state to mutation being ∼30% like that of the open state and ∼70% like that of the closed state; (c) the relationship between δ12′ amino acid residue volume, hydrophobicity or α-helical tendency, and the gating equilibrium constant of the corresponding mutants is not straightforward; however, (d) rate and equilibrium constants for the mutant series are linearly correlated (on log–log plots), which suggests that the conformational rearrangements upon mutation are mostly local and that the position of the transition state along the gating reaction coordinate is unaffected by these mutations.
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影响因子：
3.4
作者：
HORN, R;LANGE, K
通讯作者：
LANGE, K
血清胆碱激活突变的乙酰胆碱受体，导致慢通道先天性肌无力综合征。
DOI：
10.1073/pnas.96.18.10466
发表时间：
1999
影响因子：
11.1
作者：
Zhou,M;Engel,AG;Auerbach,A
通讯作者：
Auerbach,A
色氨酸合酶α亚基双突变体折叠中的协同作用。
影响因子：
2.9
作者：
Hurle,MR;Tweedy,NB;Matthews,CR
通讯作者：
Matthews,CR
乙酰胆碱受体 M2 结构域中保守亮氨酸在通道门控中的作用。
DOI：
--
发表时间：
1995
期刊：
Molecular pharmacology.
影响因子：
--
作者：
Filatov,GN;White,MM
通讯作者：
White,MM