Calcium-sensitive adenylyl cyclase/aequorin chimeras as sensitive probes for discrete modes of elevation of cytosolic calcium

Abstract

The fact that Ca 2+ -sensitive adenylyl cyclases respond to discrete, physiological elevations in cytosolic Ca 2+ ([Ca 2+ ] i ) provides a means for coordinating the actions of the two major second-messenger signaling systems. It turns out that the Ca 2+ -sensitive adenylyl cyclases are extremely discriminating in the source of the Ca 2+ to which they respond. In intact nonexcitable cells, they are regulated only by' the entry of Ca 2+ that is triggered by depletion of internal stores, so-called capacitative Ca 2+ entry (CCE). Store depletion can be triggered by hormones coupled to phosphoinositidase C, by inhibition of the Ca 2+ -reuptake pumps of the endoplasmic reticula (ER), or by ionophore insertion in the ER membranes. However, when CCE is triggered, it regulates Ca 2+ -sensitive adenylyl cyclases, whether these occur in the cells naturally or after heterologous expression. A major conceptual breakthrough came with the targeting of aequorin to discrete domains of the cell by placing an organelle-targeting sequence upstream (or downstream) of the aequorin-coding region. This allowed measurement of [Ca 2+ ] in the ER, nuclear, and mitochondrial environments. This chapter describes some of the issues involved in validating this and related constructs and their application.