CONSTRUCTION OF A FULL-LENGTH CA2-SENSITIVE ADENYLYL-CYCLASE AEQUORINCHIMERA()

Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling, To selectively probe dynamic changes in [Ca2+](i) at the pl asma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhi bitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two stage polymerase chain reaction method. The expressed adenyl yl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analys is, The chimera retained full adenylyl cyclase activity and sensitivit y to inhibition by physiological [Ca2+](i) elevation. The aequorin por tion of the chimeric construct was also capable of measuring changes i n [Ca2+] both in vitro and in vivo, When the plasma membrane-tagged ae quorin and cytosolic aequorin were compared in their measurement of [C a2+](i), they showed contrasting sensitivities depending on whether th e [Ca2+](i) originated from internal stores or capacitative entry, Thi s is the first full-length enzyme-aequorin chimera that retains the fu ll biological properties of both aequorin and a Ca2+-sensitive adenyly l 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 t his chimera is localized to the plasma membrane, it can also be used t o assess local changes in [Ca2+](i) at the plasma membrane as distinct from global changes in [Ca2+](i) within the cytosol.