Cooperative and stochastic calcium releases from multiple calcium puff sites generate calcium microdomains in intact HeLa cells

Ca²⁺ microdomains or locally restricted Ca²⁺ increases in the cell have recently been reported to regulate many essential physiological events. Ca²⁺ increases through the inositol 1,4,5-trisphosphate(IP₃)receptor(IP₃R)/Ca²⁺ release channels contribute to the formation of a class of such Ca²⁺ microdomains, which were often observed and referred to as Ca²⁺ puffs in their isolated states. In this report, we visualized IP₃-evoked Ca²⁺ microdomains in histamine-stimulated intact HeLa cells using a total internal reflection fluorescence microscope, and quantitatively characterized the spatial profile by fitting recorded images to a two-dimensional Gaussian distribution. Ca²⁺ concentration profiles were marginally spatially anisotropic, with the size increasing linearly even after the amplitude began to decline. We found the event centroid drifted with an apparent diffusion coefficient of 4.20 ± 0.50 μm²/s, which is significantly larger than those estimated for IP₃Rs. The sites of maximal Ca²⁺ increase, rather than initiation or termination sites, were detected repeatedly at the same location. These results indicate that Ca²⁺ microdomains in intact HeLa cell are generated from spatially distributed multiple IP₃R clusters or Ca²⁺ puff sites, rather than a single IP3R cluster reported in cells loaded with Ca ²⁺ buffers.