Nakahashi Y, Nelson E, Fagan K, Gonzales E, Guillou J L, Cooper D M
Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Biol Chem. 1997 Jul 18;272(29):18093-7. doi: 10.1074/jbc.272.29.18093.
Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling. To selectively probe dynamic changes in [Ca2+]i at the plasma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhibitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two-stage polymerase chain reaction method. The expressed adenylyl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analysis. The chimera retained full adenylyl cyclase activity and sensitivity to inhibition by physiological [Ca2+]i elevation. The aequorin portion of the chimeric construct was also capable of measuring changes in [Ca2+] both in vitro and in vivo. When the plasma membrane-tagged aequorin and cytosolic aequorin were compared in their measurement of [Ca2+]i, they showed contrasting sensitivities depending on whether the [Ca2+]i originated from internal stores or capacitative entry. This is the first full-length enzyme-aequorin chimera that retains the full biological properties of both aequorin and a Ca2+-sensitive adenylyl cyclase. This novel chimeric Ca2+ sensor provides the unique ability to directly report the dynamics of [Ca2+]i that regulates this Ca2+-sensitive enzyme under a variety of physiological conditions. Since this chimera is localized to the plasma membrane, it can also be used to assess local changes in [Ca2+]i at the plasma membrane as distinct from global changes in [Ca2+]i within the cytosol.
钙敏感腺苷酸环化酶是钙信号和环磷酸腺苷（cAMP）信号的关键整合者。为了选择性地探测腺苷酸环化酶所在质膜处细胞内钙离子浓度（[Ca2+]i）的动态变化，通过两步聚合酶链反应法构建了一种全长、钙抑制型VI型腺苷酸环化酶/水母发光蛋白嵌合体。通过生化分级分离和功能分析判断，所表达的腺苷酸环化酶/水母发光蛋白嵌合体被正确定位于质膜。该嵌合体保留了完整的腺苷酸环化酶活性以及对生理水平[Ca2+]i升高抑制作用的敏感性。嵌合构建体的水母发光蛋白部分在体外和体内均能够测量[Ca2+]的变化。当比较质膜标记的水母发光蛋白和胞质水母发光蛋白对[Ca2+]i的测量结果时，它们显示出不同的敏感性，这取决于[Ca2+]i是源于内部储存库还是钙池调控性钙内流。这是首个保留了水母发光蛋白和钙敏感腺苷酸环化酶完整生物学特性的全长酶 - 水母发光蛋白嵌合体。这种新型嵌合钙传感器具有独特能力，可直接报告在多种生理条件下调节这种钙敏感酶的[Ca2+]i动态变化。由于这种嵌合体定位于质膜，它还可用于评估质膜处[Ca2+]i的局部变化，这与胞质溶胶中[Ca2+]i的整体变化不同。
