Nuclear targeting of aequorin. A new approach for measuring nuclear Ca2+ concentration in intact cells.
We here describe the measurement of nuclear Ca2+ concentration ([Ca2+]n) with targeted recombinant aequorin. Two aequorin chimeras have been constructed, composed of the Ca(2+)-sensitive photoprotein and two different portions of the glucocorticoid hormone receptor (GR). The shorter chimera (nuAEQ), which contains the nuclear localization signal (NLS) NL1 of GR, but lacks its hormone binding domain, HBD, is constitutively localized in the nucleus; the longer one (nu/cytAEQ), which contains both NLSs (NL1 + NL2) and the HBS of GR, is normally localized in the cytosol, but is translocated to the nucleus upon treatment with the hormone. When localized to the nucleus, both chimeras give the same estimates of [Ca2+]n, both at rest and upon stimulation with the InsP3 generating agonist histamine. The [Ca2+]n values appear very close, both at rest and upon stimulation, to those of the cytoplasm, measured with cytosolic recombinant aequorin, suggesting that, at least in this cell model, the nuclear membrane does not represent a major barrier to the diffusion of Ca2+ ions, and that the nucleus does not regulate its [Ca2+] independently from the cytosol.
Brini
M
M
Department of Biomedical Sciences, University of Padova, Italy.
Marsault
R
R
Bastianutto
C
C
Pozzan
T
T
Rizzuto
R
R
D000331:Aequorin / Q000032:analysis* / Q000235:genetics; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics; D014162:Transfection
D011993:Recombinant Fusion Proteins; D000331:Aequorin; D002118:Calcium