Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin

EMBO J. 1993 Dec;12(12):4813-9. doi: 10.1002/j.1460-2075.1993.tb06170.x.

Abstract

Activation of nuclear transcription factors, breakdown of nuclear envelope and apoptosis represent a group of nuclear events thought to be modulated by changes in nucleoplasmic Ca2+ concentration, [Ca2+]n. Direct evidence for, or against, this possibility has been, however, difficult to obtain because measurements of [Ca2+]n are hampered by major technical problems. Here we describe a new approach for selectively monitoring Ca2+ concentrations inside the nucleus of living cells, which is based on the construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and a nuclear translocation signal derived from the rat glucocorticoid receptor. This modified aequorin (nuAEQ), stably expressed in HeLa cells, was largely confined to the nucleoplasm and thus utilized for monitoring [Ca2+]n in intact cells. No significant differences were observed between [Ca2+]n and cytosolic Ca2+ concentration ([Ca2+]i) under resting conditions. Upon stimulation of surface receptors linked to inositol-1,4,5-trisphosphate (InsP3) generation, and thus to intracellular Ca2+ signalling, the kinetics of [Ca2+]i and [Ca2+]n increases were indistinguishable. However, for the same rise in [Ca2+]i, the amplitude of [Ca2+]n increase was larger when evoked by Ca2+ mobilization from internal stores than when induced by Ca2+ influx across the plasma membrane. The functional significance of these transient nucleus-cytosol Ca2+ gradients is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aequorin / genetics
  • Aequorin / metabolism*
  • Animals
  • Blotting, Northern
  • Calcium / metabolism*
  • Cell Nucleus / metabolism*
  • Cloning, Molecular
  • HeLa Cells
  • Histamine / metabolism
  • Humans
  • Kinetics
  • Rats
  • Receptors, Glucocorticoid / genetics
  • Receptors, Glucocorticoid / metabolism
  • Recombinant Proteins / metabolism

Substances

  • Receptors, Glucocorticoid
  • Recombinant Proteins
  • Aequorin
  • Histamine
  • Calcium

Grants and funding