Construction of a full-length Ca2+-sensitive adenylyl cyclase/aequorin chimera.
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.
Nakahashi
Y
Y
Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Nelson
E
E
Fagan
K
K
Gonzales
E
E
Guillou
J L
JL
Cooper
D M
DM
D000262:Adenylyl Cyclases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000331:Aequorin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D017931:DNA Primers; D004533:Egtazic Acid / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D014162:Transfection
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D004533:Egtazic Acid; D000262:Adenylyl Cyclases; D002118:Calcium