Construction of a full-length Ca2+-sensitive adenylyl cyclase/aequorin chimera

Abstract

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.