Engineering aequorin to measure Ca2+ in defined compartments of living cells.

Aequorin is a photoprotein which emits blue light on binding, with high affinity (m 10 p~), to calcium (Ca2+) [I]. Aequorin is composed of 189-196 amino-acids, with three EF hand structures, characteristic of Ca2+ bindina sites C2.31. Aeauorin has been used as a-CaZ+ indicator in-a variety of biological systems [l]. The aim of this study was to engineer aequorin in order to measure Ca2+ changes within the endoplasmic reticulum (ER) of living cells. A consensus signal peptide sequence, known to target proteins to the [5], was added to the cDNA encoding apoaequorin. Oligonucleotide-directed sitespecific mutagenesis of one amino-acid in hand site I1 was made to lower the Ca2+ affinity of aequorin, for the "high" Ca2+ likely to be present in the ER. Addition of the T7 RNA polymerase ER