A quantitative description of KcsA gating I: macroscopic currents.
A quantitative description of KcsA gating I: macroscopic currents.
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DOI：
10.1085/jgp.200709843
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发表时间：
2007-11
影响因子：
3.8
通讯作者：
Perozo, Eduardo
中科院分区：
文献类型：
--
作者：
Chakrapani, Sudha;Cordero-Morales, Julio F;Perozo, Eduardo
原核生物的K+通道KcsA由细胞内质子激活，其门控由跨膜电压调节。通常情况下，KcsA功能已经研究了稳态条件下，使用宏观Rb+通量实验和单通道电流测量。这些研究提供了有限的见解KcsA的门控动力学，由于其低的开放概率，在补丁中的通道数量的不确定性，和一个非常强大的内在动力学变异性。在这项工作中，我们进行了详细的分析KcsA门控在非稳态条件下，通过检查pH值和电压的影响，激活，失活，和缓慢失活门控事件。我们发现，激活和失活门控的KcsA主要是由pH值调制的电压没有显着的影响。活化门控显示S形pH依赖性，pKa为1.44.2，希尔系数为1.442。在质子的持续存在下，KcsA的电导随时间衰减。该失活过程与pH无关，但受电压和渗透离子性质的调节。从失活的恢复通过失活发生，并且似乎也是电压依赖性的。我们进一步发现，KcsA的失活不完全是开放传导通道的特性，但也可以发生在部分“激活”的封闭状态。从这些预开放封闭状态的失活过程的开始和恢复的时间过程似乎与开放状态失活不同，表明存在具有不同动力学途径的多个失活状态。这一信息已被分析一起详细研究KcsA单通道行为（在随附的文件）的框架内的动力学模型。总之，我们的数据构成了KcsA门控的第一个定量描述。
The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.