Gain-of-function mutations reveal expanded intermediate states and a sequential action of two gates in MscL.
Gain-of-function mutations reveal expanded intermediate states and a sequential action of two gates in MscL.
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DOI：
10.1085/jgp.200409118
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发表时间：
2005-02
期刊：
影响因子：
--
通讯作者：
Sukharev S
中科院分区：
文献类型：
--
作者：
Anishkin A;Chiang CS;Sukharev S
大机械敏感通道 MscL 中的张力驱动的门控转换通过可检测的中间电导状态进行。在主疏水门中具有极性或带电取代的功能获得（GOF）突变体显示出亚导态模式的改变，提供了有关门控中间体的有价值的信息。在这里，我们对几种 GOF 突变体进行热力学分析，以阐明过渡途径中低传导构象的性质和位置。与野生型 (WT) MscL 主要占据具有非常短暂的亚导状态的闭合和完全开放状态不同，温和的 V23T GOF 突变体经常访问多个短暂的亚导状态。严重突变体 V23D 和 G22N 按顺序打开：关闭 (C) → 低导电亚状态 (S) → 打开 (O)，第一个亚转变发生在较低张力下。对平衡态占据作为膜张力函数的分析表明，WT MscL 中的 C→S 亚转变仅与较小的电导增量相关，但与最大的面内膨胀和自由能变化相关。 GOF 取代通过减少 C 和 S 状态之间的面积 (ΔA) 和能量 (ΔE) 变化来强烈影响第一次亚转变，这与突变的严重程度相当。 GOF 突变体还表现出与第二次 (S→O) 亚转变相关的相当大的 ΔE，但 ΔA 与 WT 相似。面积变化表明 GOF 突变体的闭合构象在物理上已预扩展。通道关闭 (k off) 的速率常数的张力依赖性预测了 WT 和 GOF MscL 能量区域剖面上限速势垒的不同位置。数据支持双门机制，其中第一个亚转变（C→S）可以被视为中央（M1）门的打开，导致膨胀的充满水的“泄漏”构象。极性 GOF 取代对这一步骤的大力促进表明，与 WT MscL 中孔的水合相关的 M1 螺旋的分离是打开的主要能量障碍。具有稳定 S1 门的突变体表现出从低导电亚状态到完全开放状态的转变受阻，而 S1-M1 连接子的延伸导致反向 O→S 转变的可能性更高。这些数据强烈表明，第二个子转变 (S→O) 中的大部分电导增益是通过 NH2 末端 (S1) 栅极的打开而发生的，并且连接器是 M1 和 S1 栅极之间的耦合元件。
The tension-driven gating transition in the large mechanosensitive channel MscL proceeds through detectable states of intermediate conductance. Gain-of-function (GOF) mutants with polar or charged substitutions in the main hydrophobic gate display altered patterns of subconducting states, providing valuable information about gating intermediates. Here we present thermodynamic analysis of several GOF mutants to clarify the nature and position of low-conducting conformations in the transition pathway. Unlike wild-type (WT) MscL, which predominantly occupies the closed and fully open states with very brief substates, the mild V23T GOF mutant frequently visits a multitude of short-lived subconducting states. Severe mutants V23D and G22N open in sequence: closed (C) → low-conducting substate (S) → open (O), with the first subtransition occurring at lower tensions. Analyses of equilibrium state occupancies as functions of membrane tension show that the C→S subtransition in WT MscL is associated with only a minor conductance increment, but the largest in-plane expansion and free energy change. The GOF substitutions strongly affect the first subtransition by reducing area (ΔA) and energy (ΔE) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger ΔE associated with the second (S→O) subtransition, but a ΔA similar to WT. The area changes indicate that closed conformations of GOF mutants are physically preexpanded. The tension dependencies of rate constants for channel closure (k off) predict different positions of rate-limiting barriers on the energy-area profiles for WT and GOF MscL. The data support the two-gate mechanism in which the first subtransition (C→S) can be viewed as opening of the central (M1) gate, resulting in an expanded water-filled “leaky” conformation. Strong facilitation of this step by polar GOF substitutions suggests that separation of M1 helices associated with hydration of the pore in WT MscL is the major energetic barrier for opening. Mutants with a stabilized S1 gate demonstrate impeded transitions from low-conducting substates to the fully open state, whereas extensions of S1–M1 linkers result in a much higher probability of reverse O→S transitions. These data strongly suggest that the bulk of conductance gain in the second subtransition (S→O) occurs through the opening of the NH2-terminal (S1) gate and the linkers are coupling elements between the M1 and S1 gates.
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