Dynamic and quantitative Ca2+ measurements using improved cameleons
Dynamic and quantitative Ca2+ measurements using improved cameleons
复制标题
DOI：
10.1073/pnas.96.5.2135
复制
发表时间：
1999-03-02
影响因子：
11.1
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Miyawaki, A;Griesbeck, O;Tsien, RY
Cameleon是基于绿色荧光蛋白变体和钙调蛋白（CaM）的遗传编码的Ca2+荧光指示剂。由于cameleons可以有针对性的遗传和成像的单光子或双光子激发显微镜，他们提供了很大的承诺，监测钙离子在整个生物体，组织，细胞器，和亚微观环境中的测量是以前不可能的。然而，原来的骆驼遭受显着的pH干扰，其Ca2+缓冲和交叉反应性与内源性钙调素信号转导途径是未知的。我们现在通过将突变V68 L和Q69 K引入受体黄绿色荧光蛋白中，大大降低了骆驼的pH敏感性。由此产生的新cameleons允许Ca2+测量，尽管显着的胞质酸化。当Ca2+升高时，Cameleon中融合在一起的CaM和CaM结合肽主要彼此相互作用，而不是与游离CaM和CaM依赖性酶相互作用。因此，如果cameleons过表达，主要的影响可能是不可避免的增加钙缓冲，而不是特定的干扰钙调蛋白依赖性信号。
Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+-buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.