The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference.
The secretory-pathway Ca(2+)-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca(2+) pump found mostly in the Golgi compartment. We have explored the contribution of this Ca(2+) pump to cytosolic Ca(2+) signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca(2+) concentration in the lumen of the Golgi apparatus by specifically targeting the Ca(2+)-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca(2+) uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca(2+) spiking when stimulated with histamine, indicating that the SPCA1-containing Ca(2+) store was not absolutely needed to set up these oscillations. However, baseline Ca(2+) oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca(2+) signal in HeLa cells.
Van Baelen
Kurt
K
Laboratorium voor Fysiologie, K.U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000, Leuven, Belgium.
Vanoevelen
Jo
J
Callewaert
Geert
G
Parys
Jan B
JB
De Smedt
Humbert
H
Raeymaekers
Luc
L
Rizzuto
Rosario
R
Missiaen
Ludwig
L
Wuytack
Frank
F
D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019284:Thapsigargin; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium