Microscopic properties of elementary Ca2+ release sites in non-excitable cells.

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. 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. 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.