Miyawaki A, Griesbeck O, Heim R, Tsien R Y
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA.
Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2135-40. doi: 10.1073/pnas.96.5.2135.
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.
钙调荧光蛋白是基于绿色荧光蛋白变体和钙调蛋白（CaM）的基因编码钙（Ca2+）荧光指示剂。由于钙调荧光蛋白可通过基因靶向，并能用单光子或双光子激发显微镜成像，因此在监测完整生物体、组织、细胞器以及以前无法进行测量的亚微观环境中的Ca2+方面具有巨大潜力。然而，最初的钙调荧光蛋白存在显著的pH干扰，并且其Ca2+缓冲能力以及与内源性CaM信号通路的交叉反应性尚未得到表征。我们现在通过在受体黄绿色荧光蛋白中引入V68L和Q69K突变，大大降低了钙调荧光蛋白的pH敏感性。由此产生的新型钙调荧光蛋白即使在胞质显著酸化的情况下也能进行Ca2+测量。当Ca2+升高时，在钙调荧光蛋白中融合在一起的CaM和CaM结合肽主要相互作用，而不是与游离的CaM和CaM依赖性酶相互作用。因此，如果过度表达钙调荧光蛋白，主要影响可能是不可避免的Ca2+缓冲增加，而不是CaM依赖性信号传导的特异性扰动。
