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© The Rockefeller University Press, 0022-1295/1999//239/ $5.00
Journal of General Physiology, Volume 113, Number 2, 1999

Article

Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

Carsten Strübing* and David E. Clapham{ddagger}

From the * Department of Neurobiology, Harvard Medical School, Howard Hughes Medical Institute, and {ddagger} Division of Cardiovascular Research, Children's Hospital Boston, Boston, Massachusetts 02115

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller ({approx}10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.

Key Words: nuclear transport • nuclear pore complex • Ca2+ store depletion • green fluorescent protein

Address correspondence to David E. Clapham, Division of Cardiovascular Research, Children's Hospital Boston, Enders Building, P.O. Box EN-306, 320 Longwood Avenue, Boston, MA 02115. Fax: 617-730-0692; E-mail: clapham{at}rascal.med.harvard.edu

Abbreviations: AM, acetoxymethyl ester; Aniso, anisomycin; BAPTA, 1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid; Dex, dexamethasone; ER, endoplasmic reticulum; GFP, green fluorescent protein; GR, glucocorticoid receptor; InsP3, inositol- (1,4,5) trisphosphate; MK2, MAP kinase–activated protein kinase 2; NF/CF, ratio of nuclear/cytoplasmic fluorescence intensity; NLS, nuclear localization sequence; NPC, nuclear pore complex; TPEN, tetrakis-[2-pyridylmethyl]-ethylenediamine


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