The "dashpot" mechanism of stretch-dependent gating in MscS.
The "dashpot" mechanism of stretch-dependent gating in MscS.
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DOI：
10.1085/jgp.200409198
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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：
Akitake B;Anishkin A;Sukharev S
小电导机械敏感通道（MscS）的晶体结构一直是寻找门控机制的宝贵工具，但该通道的许多功能方面仍悬而未决。在这里，我们在三重突变体（mscL - ，mscS - ，mscK - ）背景下描述了大肠杆菌原生质球中MscS的门控。我们使用压力钳装置和实验室开发的软件直接从电流和压力的双通道记录生成剂量反应曲线。与之前的出版物相比，我们发现 MScS 表现出基本上与电压无关的张力激活，但同时在去极化条件下表现出强烈的电压依赖性失活。在不同电压下的饱和压力斜坡下获得的 MScS 激活曲线给出了关闭到打开转变的能量、面积和门控电荷的估计值，分别为 24 kT、18 nm2 和 +0.8。 K+ 和 Na+ 缓冲液中的激活和失活特征相似。也许 MscS 门控最显着和最有趣的特性是对压力施加速率的强烈依赖。贴片经受从 2.7 到 240 mmHg/s 的各种压力梯度，显示出几乎与速率无关的激活中点。然而，当缓慢施加压力时，特别是在去极化移液器电压下，所产生的通道活性显着降低。看来，MscS 更喜欢对突然的刺激做出全面反应，但设法忽略那些缓慢施加的刺激，就好像门通过速度敏感的“缓冲器”连接到张力传递元件一样。当斜坡较慢时，通道在通过低于激活中点的狭窄压力区域时会失活。这种“倾倒”缓慢施加的力的特性在细胞补水逐渐发生并且不希望释放渗透剂的环境情况下可能很重要。 MScS 通常通过去极化电压有利于的亚导状态进入失活状态。失活率随着去极化呈指数增加。基于这些结果，我们提出了一种动力学方案和门控机制，以解释在可用结构信息框架中观察到的现象学。
The crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. Here we characterized the gating of MscS in Escherichia coli spheroplasts in a triple mutant (mscL − , mscS − , mscK −) background. We used a pressure clamp apparatus along with software developed in-lab to generate dose–response curves directly from two-channel recordings of current and pressure. In contrast to previous publications, we found that MscS exhibits essentially voltage-independent activation by tension, but at the same time strong voltage-dependent inactivation under depolarizing conditions. The MscS activation curves obtained under saturating ramps of pressure, at different voltages, gave estimates for the energy, area, and gating charge for the closed-to-open transition as 24 kT, 18 nm2, and +0.8, respectively. The character of activation and inactivation was similar in both K+ and Na+ buffers. Perhaps the most salient and intriguing property of MscS gating was a strong dependence on the rate of pressure application. Patches subjected to various pressure ramps from 2.7 to 240 mmHg/s revealed a midpoint of activation almost independent of rate. However, the resultant channel activity was dramatically lower when pressure was applied slowly, especially at depolarizing pipette voltages. It appears that MscS prefers to respond in full to abrupt stimuli but manages to ignore those applied slowly, as if the gate were connected to the tension-transmitting element via a velocity-sensitive “dashpot.” With slower ramps, channels inactivate during the passage through a narrow region of pressures below the activation midpoint. This property of “dumping” a slowly applied force may be important in environmental situations where rehydration of cells occurs gradually and release of osmolytes is not desirable. MscS often enters the inactivated state through subconducting states favored by depolarizing voltage. The inactivation rate increases exponentially with depolarization. Based on these results we propose a kinetic scheme and gating mechanism to account for the observed phenomenology in the framework of available structural information.
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