Microscopic properties of elementary Ca2+ release sites in non-excitable cells.
BACKGROUND
Elementary Ca2+ signals, such as 'Ca2+ puffs', that arise from the activation of clusters of inositol 1 ,4,5,-trisphosphate (InsP3) receptors are the building blocks for local and global Ca2+ signalling. We previously found that one, or a few, Ca2+ puff sites within agonist-stimulated cells act as 'pacemakers' to initiate global Ca2+ waves. The factors that distinguish these pacemaker Ca2+ puff sites from the other Ca2+ release sites that simply participate in Ca2+ wave propagation are unknown.


RESULTS
The spatiotemporal properties of Ca2+ puffs were investigated using confocal microscopy of fluo3-loaded HeLa cells. The same pacemaker Ca2+ puff sites were activated during stimulation of cells with different agonists. The majority of agonist-stimulated pacemaker Ca2+ puffs originated in a perinuclear location. The positions of such Ca2+ puff sites were stable for up to 2 hours, and were not affected by disruption of the actin cytoskeleton. A similar perinuclear distribution of Ca2+ puff sites was also observed when InsP3 receptors were directly stimulated with thimerosal or membrane-permeant InsP3 esters. Immunostaining indicated that the perinuclear position of pacemaker Ca2+ puffs was not due to the localised expression of InsP3 receptors.


CONCLUSIONS
The pacemaker Ca2+ puff sites that initiate Ca2+ responses are temporally and spatially stable within cells. These Ca2+ release sites are distinguished from their neighbours by an intrinsically higher InsP3 sensitivity.
Thomas
D
D
Laboratory of Molecular Signalling, The Babraham Institute, Cambridge, UK.
Lipp
P
P
Tovey
S C
SC
Berridge
M J
MJ
Li
W
W
Tsien
R Y
RY
Bootman
M D
MD
D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D017607:Aluminum Compounds / Q000494:pharmacology; D015220:Calcium Channels / Q000032:analysis / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D005459:Fluorides / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010507:Periodicity; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000502:physiology*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013849:Thimerosal / Q000494:pharmacology
D017607:Aluminum Compounds; D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015638:Cytochalasin D; D013849:Thimerosal; D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D000109:Acetylcholine; D005459:Fluorides; C032311:aluminum fluoride