A quantitative description of KcsA gating II: single-channel currents.
A quantitative description of KcsA gating II: single-channel currents.
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DOI：
10.1085/jgp.200709844
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发表时间：
2007-11
期刊：
影响因子：
--
通讯作者：
Perozo E
中科院分区：
文献类型：
--
作者：
Chakrapani S;Cordero-Morales JF;Perozo E
在纯化的脂质体重构制剂中，在单通道水平上研究了质子激活的 WT KcsA 和非失活的 E71A 突变体的动力学转变。在非稳态和稳态条件下使用膜片钳技术记录单通道电流。最大似然分析表明，酸性 pH 值的关键影响是增加爆发频率，而不影响爆发内打开和关闭停留时间，这与宏观电流的发现一致，即质子促进激活而不对失活产生显着影响。然而，在 pH 值稳定的条件下，电压不仅会改变突发频率，还会影响其特性，例如闪烁频率以及关闭和打开状态的停留时间。如果电压调节连接开放状态和失活状态的通路，则这是可以预料的。打开后，KcsA 可以进入至少两种不属于激活途径的关闭状态。发现这些关闭状态的频率和持续时间与电压有关，因此这些可能代表短暂的失活状态。 WT KcsA 的单通道记录也显示出亚电导状态存在的不同倾向。这些状态发生的概率并未显示出电压或 pH 值的明显调节，其起源仍不清楚，也是进一步研究的重点。提出了一个动力学模型来描述 KcsA 中的门控事件，概括其宏观和单通道行为。该模型受到本工作中提出的单通道分析以及前一篇论文中宏观电流数据的限制。
The kinetic transitions of proton-activated WT KcsA and the noninactivating E71A mutant were studied at the single-channel level in purified, liposome-reconstituted preparations. Single-channel currents were recorded using patch-clamp techniques under nonstationary and steady-state conditions. Maximum-likelihood analyses reveal that the key influence of acidic pH is to increase the frequency of bursting without an effect on the intraburst open and closed dwell times, consistent with the finding from macroscopic currents that protons promote activation without a significant effect on inactivation. However, in steady-conditions of pH, voltage not only alters the burst frequency but also affects their properties, such as the frequency of the flickers and the dwell times of the closed and open states. This is to be expected if voltage modulates pathways connecting open and inactivated states. Upon opening, KcsA can enter at least two closed states that are not part of the activation pathway. The frequency and duration of these closed states was found to be voltage dependent and therefore these are likely to represent short-lived inactivated states. Single-channel recordings of WT KcsA also show varying propensity for the presence of subconductance states. The probability of occurrence of these states did not show clear modulation by voltage or pH and their origin remains unclear and a focus for further investigation. A kinetic model is proposed to describe the gating events in KcsA that recapitulates its macroscopic and single-channel behavior. The model has been constrained by the single-channel analyses presented in this work along with data from macroscopic currents in the preceding paper.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
DOI：
10.1085/jgp.200609638
发表时间：
2006-11
期刊：
The Journal of general physiology
影响因子：
--
作者：
Blunck R;Cordero-Morales JF;Cuello LG;Perozo E;Bezanilla F
通讯作者：
Bezanilla F
影响因子：
5.5
作者：
AUERBACH, A;LINGLE, CJ
通讯作者：
LINGLE, CJ
影响因子：
2.9
作者：
Cortes, DM;Perozo, E
通讯作者：
Perozo, E
影响因子：
2.9
作者：
Gross, A;Columbus, L;Hubbell, WL
通讯作者：
Hubbell, WL