Visualization of inositol 1,4,5-trisphosphate receptor type III with green fluorescent protein in living cells.
Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel that releases Ca2+ into the cytosol and its subcellular distribution is believed to have significant effects on Ca2+ signalling. We constructed a plasmid vector containing full-length rat type 3 IP3R linked to GFP (GFP-IP3R) for expression in mammalian cells. Western blot analyses revealed that the expressed fusion protein contained both GFP and full-length type 3 IP3R. Fluorescence confocal microscopy showed that the fluorescence of GFP-IP3R3 was distributed to reticular network structures, even after cell permeabilization with saponin. We further visualized intracellular membranes with DiOC6, a vital fluorescent marker for intracellular membranes, and provide evidence that the distribution of GFP-IP3R3 overlaps with the distribution of the endoplasmic reticulum. Our results indicate that GFP-IP3R3 can be used to visualize IP3R in living cells, and pave the way for subsequent mutational and functional studies.
Morita
T
T
Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan.
Tanimura
A
A
Nezu
A
A
Tojyo
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D015220:Calcium Channels / Q000032:analysis* / Q000235:genetics; D004274:DNA, Recombinant; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008164:Luminescent Proteins / Q000235:genetics*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D004274:DNA, Recombinant; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008164:Luminescent Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins