Endogenous signalling pathways and caged-IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites

The building blocks for intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of a small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs in human embryonic kidney 293 cells evoked by photolysis of caged-IP3 or activation of endogenous muscarinic receptors with carbachol. Ca2+ puffs evoked by carbachol initiated at an estimated 65 ± 7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged-IP3 evoked Ca2+ puffs at a similar number of sites (100 ± 35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential challenges with carbachol and photolysis of caged-IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites, the sites become more likely to fire as the IP3 concentration increases, and there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.This work was supported by the Wellcome Trust [grant number 101844], by a studentship to M.V.K. from the Cambridge Overseas Trust, and by awards to M.V.K. from St. John's College, Cambridge and the Cambridge Philosophical Society. Deposited in PMC for immediate release

A Statistical View on Calcium Oscillations.

Transient rises and falls of the intracellular calcium concentration have been observed in numerous cell types and under a plethora of conditions. There is now a growing body of evidence that these whole-cell calcium oscillations are stochastic, which poses a significant challenge for modelling. In this review, we take a closer look at recently developed statistical approaches to calcium oscillations. These models describe the timing of whole-cell calcium spikes, yet their parametrisations reflect subcellular processes. We show how non-stationary calcium spike sequences, which e.g. occur during slow depletion of intracellular calcium stores or in the presence of time-dependent stimulation, can be analysed with the help of so-called intensity functions. By utilising Bayesian concepts, we demonstrate how values of key parameters of the statistical model can be inferred from single cell calcium spike sequences and illustrate what information whole-cell statistical models can provide about the subcellular mechanistic processes that drive calcium oscillations. In particular, we find that the interspike interval distribution of HEK293 cells under constant stimulation is captured by a Gamma distribution