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Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase
Debbie Willoughby, Sebastian Wachten, Nanako Masada, Dermot M. F. Cooper


Ca2+-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca2+ and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca2+ entry (CCE) over other modes of Ca2+ increase. To directly address the possibility that these ACs reside in discrete Ca2+ microdomains, we tethered a Ca2+ sensor, GCaMP2, to the N-terminus of Ca2+-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca2+-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca2+ rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca2+ fluxes during Ca2+ mobilization and non-specific Ca2+ entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca2+-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca2+ signals seen throughout the cytosol. By contrast, a unique Ca2+ microdomain surrounds AC8 that promotes selectivity for Ca2+ signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca2+ signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.


  • Supplementary material available online at

  • Many thanks to Michael Kotlikoff (Cornell University, New York, NY) and Martin Lohse (Würzberg University, Germany) for the kind gifts of plasmid DNA encoding GCaMP2 and Epac2-camps, respectively. This work was funded by The Wellcome Trust (RG 31760). DMFC is a Royal Society Wolfson Research Fellow. Deposited in PMC for release after 6 months.

  • Accepted November 3, 2009.
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