Structural implications of fluorescence quenching in the Shaker K+ channel.
Structural implications of fluorescence quenching in the Shaker K+ channel.
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DOI：
10.1085/jgp.112.4.391
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发表时间：
1998-10
期刊：
影响因子：
--
通讯作者：
Bezanilla F
中科院分区：
文献类型：
--
作者：
Cha A;Bezanilla F
当连接到不导电的（W434F） Shaker钾通道S4段附近的特定位点时，荧光探针四甲基罗达明酰亚胺经历与电压传感器运动相关的电压依赖性强度变化（Mannuzzu, l.m.， M.M. Moronne和E.Y. Isacoff. 1996）。科学。271:213 - 216;Cha， A.和F. Bezanilla. 1997。神经元。19:1127 - 1140)。这种电压依赖性荧光猝灭的特性在具有不同孔取代（T449Y）的通道的导电版本中是不同的。用四乙基铵或agitoxin阻断T449Y结构体的孔，可以去除与电压依赖性相关的荧光成分，但不能去除离子激活动力学。这种孔介导的S4段附近荧光猝灭的调制表明，荧光团受到外部孔状态的影响。此外，这种调制可能反映了与通道打开相关的构象变化，而这种变化是由四乙基铵或agitoxin阻止的。pH滴定、碰撞猝灭剂和各向异性的研究表明，附着在S4片段附近残基上的荧光团受到附近蛋白质区域的约束。S4段附近的荧光猝灭机制既不涉及荧光团的重定向，也不涉及电压依赖的激发位移，与S2段附近观察到的猝灭机制不同。综上所述，这些结果表明，S4片段的细胞外部分位于水蛋白前庭，并受外孔状态的影响。
When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213–216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127–1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.
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影响因子：
16.2
作者：
Aggarwal, SK;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
16.2
作者：
Larsson, HP;Baker, OS;Isacoff, EY
通讯作者：
Isacoff, EY
影响因子：
56.9
作者：
MACKINNON, R;YELLEN, G
通讯作者：
YELLEN, G
影响因子：
3.4
作者：
ANDREEV, OA;ANDREEVA, AL;BOREJDO, J
通讯作者：
BOREJDO, J
影响因子：
2.9
作者：
COELHOSAMPAIO, T;VOSS, EW
通讯作者：
VOSS, EW