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

Yoshitsugu Nakahashi,Eric Nelson,Kent Fagan,Dermot M.F Cooper,Elizabeth Gonzales,Jean-Louis Guillou

doi:10.1074/jbc.272.29.18093

Yoshitsugu Nakahashi, Eric Nelson + Show 4 more

Open Access

https://doi.org/10.1074/jbc.272.29.18093

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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.

Highlights

The jellyfish protein aequorin with its co-factor, coelenterazine, has been used for over 3 decades to measure cytosolic calcium ([Ca2ϩ]i)1 [1]
The regulation by Ca2ϩ is due predominantly to capacitative Ca2ϩ entry; Ca2ϩ released from internal stores or nonspecific entry of Ca2ϩ via ionophore is unable to regulate Ca2ϩ-sensitive adenylyl cyclases [11, 12]
Somewhat paradoxical is the fact that measurements of global cytosolic [Ca2ϩ]i, using the fluorescent Ca2ϩ-indicator fura-2, reveal that Ca2ϩ released from internal stores or nonspecific entry of Ca2ϩ via ionophore reports far higher levels of [Ca2ϩ]i than are achieved by capacitative Ca2ϩ entry [11, 12]

Summary

Introduction

The jellyfish protein aequorin with its co-factor, coelenterazine, has been used for over 3 decades to measure cytosolic calcium ([Ca2ϩ]i)1 [1]. Were introduced to the bathing medium to initiate capacitative Ca2ϩ entry and the cAMP response measured, it was clear that both ACVI- and ACVI/AEQ-transfected cells showed the same (approximately 40%) maximal inhibition (Fig. 3B).

Results

Conclusion