Ca2+-dependent and -independent mechanisms of calmodulin nuclear translocation.
Translocation from the cytosol to the nucleus is a major response by calmodulin (CaM) to stimulation of cells by Ca2+. However, the mechanisms involved in this process are still controversial and both passive and facilitated diffusion have been put forward. We tested nuclear translocation mechanisms in electroporated HeLa cells, rat cortical neurons and glial cells using novel calmodulin and inhibitor peptide probes and confocal microscopy. Passive diffusion of calmodulin across the nuclear membrane was measured in conditions in which facilitated transport was blocked and was compared to that of a similarly sized fluorescein-labeled dextran. Wheat germ agglutinin, which blocks facilitated transport but not passive diffusion, inhibited the nuclear entry of both wild-type and Ca2+-binding-deficient mutant calmodulin both in low and elevated [Ca2+]. Ca2+-dependent nuclear translocation was prevented by a membrane-permeant CaM inhibitor, the mTrp peptide, which indicated that it was specific to Ca2+/CaM. Diffusion of free CaM and Ca2+/CaM was considerably slower than the observed nuclear translocation by facilitated transport. Our data show that the majority of CaM nuclear entry occurred by facilitated mechanisms in all cell types examined, in part by a Ca2+-independent and in part by a Ca2+-dependent translocation mechanism.
Thorogate
Richard
R
Department of Basic Medical Sciences, St Georges Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK.
Török
Katalin
K
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000831:Animals, Newborn; D001692:Biological Transport; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D018274:Electroporation; D019793:Fluorescein / Q000494:pharmacology; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D018613:Microscopy, Confocal; D009457:Neuroglia / Q000166:cytology; D009474:Neurons / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014909:Wheat Germ Agglutinins / Q000494:pharmacology
D002147:Calmodulin; D018076:DNA, Complementary; D003911:Dextrans; D010455:Peptides; D014909:Wheat Germ Agglutinins; D010727:Phosphoric Diester Hydrolases; D014357:Trypsin; D002118:Calcium; D019793:Fluorescein