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.