Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction.
Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction.
复制标题
DOI：
10.1085/jgp.200609638
复制
发表时间：
2006-11
期刊：
影响因子：
--
通讯作者：
Bezanilla F
中科院分区：
文献类型：
--
作者：
Blunck R;Cordero-Morales JF;Cuello LG;Perozo E;Bezanilla F
封闭的KcsA通道结构揭示了跨膜螺旋的胞质末端的交叉阻断了渗透途径。通常认为，在通道打开期间，该螺旋束交叉必须加宽，以便能够进入内腔。在这里，我们解决的问题，是否打开的内门是足够的离子传导，或者如果第二个门，位于其他地方，可能会中断离子流。我们使用荧光寿命测量KcsA通道标记的四甲基罗丹明在TM 2的C-末端的残基报告的开放较低的孔区域。我们发现两个群体的通道具有不同的荧光寿命，其相对分布同意的开放概率的通道。发现具有开放束交叉的通道的绝对比例太高，无法解释KcsA-WT通道的低开放概率。我们发现了相同的分布在WT通道之间的开放和封闭的束交叉两个KcsA突变体，A73 E和E71 A，这显着增加开放的概率在低pH值。这两个结果强烈表明，在离子渗透途径中存在第二个门。突变A73 E和E71 A的位置表明第二个门可能是选择性过滤器，其在稳态条件下处于失活状态。由于在KcsA-WT中观察到的长关闭时间在KcsA-A73 E或-E71 A中不存在，我们提出KcsA-WT主要保持在具有开放束交叉但关闭（失活）第二门的状态，而突变A73 E和E71 A急剧降低进入失活状态的趋势，因此，第二门在稳态时主要打开。光学监测束交叉的开口的能力使得能够在通道起作用时直接记录孔螺旋的运动。
The closed KcsA channel structure revealed a crossing of the cytosolic ends of the transmembrane helices blocking the permeation pathway. It is generally agreed that during channel opening this helical bundle crossing has to widen in order to enable access to the inner cavity. Here, we address the question of whether the opening of the inner gate is sufficient for ion conduction, or if a second gate, located elsewhere, may interrupt the ion flow. We used fluorescence lifetime measurements on KcsA channels labeled with tetramethylrhodamine at residues in the C-terminal end of TM2 to report on the opening of the lower pore region. We found two populations of channels with different fluorescence lifetimes, whose relative distribution agrees with the open probability of the channel. The absolute fraction of channels found with an open bundle crossing is too high to explain the low open probability of the KcsA-WT channel. We found the same distribution as in the WT channel between open and closed bundle crossing for two KcsA mutants, A73E and E71A, which significantly increase open probability at low pH. These two results strongly suggest that a second gate in the ion permeation pathway exists. The location of the mutations A73E and E71A suggests that the second gate may be the selectivity filter, which resides in an inactivated state under steady-state conditions. Since the long closed times observed in KcsA-WT are not present in KcsA-A73E or -E71A, we propose that KcsA-WT remains predominantly in a state with an open bundle crossing but closed (inactivated) second gate, while the mutations A73E and E71A sharply decrease the tendency to enter in the inactivated state, and as a consequence, the second gate is predominantly open at steady state. The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning.
登录
查看更多内容
影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
全长KCSA的分子结构：细胞质结构域在离子渗透和激活门口中的作用。
影响因子：
3.8
作者：
Cortes, DM;Cuello, LG;Perozo, E
通讯作者：
Perozo, E
影响因子：
2.9
作者：
Cortes, DM;Perozo, E
通讯作者：
Perozo, E
DOI：
10.1085/jgp.114.4.551
发表时间：
1999-10
期刊：
The Journal of general physiology
影响因子：
--
作者：
Heginbotham L;LeMasurier M;Kolmakova-Partensky L;Miller C
通讯作者：
Miller C
影响因子：
64.8
作者：
Jiang, YX;Ruta, V;MacKinnon, R
通讯作者：
MacKinnon, R