Selective zinc sensor molecules with various affinities for Zn2+, revealing dynamics and regional distribution of synaptically released Zn2+ in hippocampal slices
Selective zinc sensor molecules with various affinities for Zn2+, revealing dynamics and regional distribution of synaptically released Zn2+ in hippocampal slices
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DOI：
10.1021/ja050301e
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发表时间：
2005-07-27
影响因子：
15
通讯作者：
Nagano, T
中科院分区：
文献类型：
--
作者：
Komatsu, K;Kikuchi, K;Nagano, T
我们已经开发了一系列的荧光Zn 2+传感器分子与不同的亲和力Zn 2+，因为生物Zn 2+浓度变化范围很广，从亚纳摩尔到毫摩尔。新传感器的Kd值在10（-8）-10（-4）M范围内，而ZnAF-2的Kd值为2.7 nM。它们在其他生物学上重要的金属离子如钙或镁的存在下不发荧光，并且它们可以在100 ms内检测到Zn 2+。在培养的细胞中，ZnAF-2的荧光强度在低Zn 2+浓度下饱和，而ZnAF-3（Kd = 0.79 μ M）的荧光强度即使在相对高的Zn 2+浓度下也不饱和。在海马切片中，我们测量了高钾诱导的去极化引起的突触释放Zn 2+。ZnAF-2在齿状回（DG）、CA 3和CA 1中显示出相似的荧光增加水平，这是不可区分的。然而，ZnAF-3仅在DG中显示荧光增加。因此，通过使用传感器分子的组合，首次证明DG中释放的Zn 2+浓度高于CA 3或CA 1，并且我们可以容易地在宽范围内可视化Zn 2+浓度。我们相信，ZnAF家族成员的各种组合的使用将为生物应用中Zn 2+的荧光显微成像提供前所未有的多功能性。
We have developed a series of fluorescent Zn2+ sensor molecules with distinct affinities for Zn2+, because biological Zn2+ concentrations vary over a wide range from sub-nanomolar to millimolar. The new sensors have Kd values in the range of 10(-8)-10(-4) M, compared with 2.7 nM for ZnAF-2. They do not fluoresce in the presence of other biologically important metal ions such as calcium or magnesium, and they can detect Zn2+ within 100 ms. In cultured cells, the fluorescence intensity of ZnAF-2 was saturated at low Zn2+ concentration, while that of ZnAF-3 (K-d = 0.79 mu M) was not saturated even at relatively high Zn2+ concentrations. In hippocampal slices, we measured synaptic release of Zn2+ in response to high-potassium-induced depolarization. ZnAF-2 showed similar levels of fluorescence increase in dentate gyrus (DG), CA3 and CA1, which were indistinguishable. However, ZnAF-3 showed a fluorescence increase only in DG. Thus, by using a combination of sensor molecules, it was demonstrated for the first time that a higher Zn2+ concentration is released in DG than in CA3 or CA1 and that we can easily visualize Zn2+ concentration over a wide range. We believe that the use of various combinations of ZnAF family members will offer unprecedented versatility for fluorescence-microscopic imaging of Zn2+ in biological applications.