The elementary release events underlying inositol 1,4,5‐trisphosphate (InsP3)‐mediated calcium signalling were investigated in Xenopus oocytes by means of high‐resolution confocal linescan imaging together with flash photolysis of caged InsP3. Weak photolysis flashes evoked localized, transient calcium signals that arose at specific sites following random latencies of up to several seconds. The duration, spatial spread and amplitude of these elementary events varied widely. Event durations (at half‐maximal amplitude) were distributed exponentially between about 100 and 600 ms. Fluorescence magnitudes (F/F0 of Oregon Green 488 BAPTA‐1) showed a skewed distribution with a peak at about 1.5 and a tail extending as high as 3.5. Individual release sites exhibited both small events (blips) and large events (puffs). The spatiotemporal distribution of calcium signals during puffs was consistent with calcium diffusion from a point source (< a few hundred nanometres), rather than with propagation of a microscopic calcium wave. Estimates of the calcium flux associated with individual events were made by integrating fluorescence profiles along the scan line in three dimensions to derive the ‘signal mass’ at each time point. The smallest resolved events corresponded to liberation of < 2 × 10−20 mol Ca2+, and large events to about 2 × 10−18 mol Ca2+. The rise of signal mass was more prolonged than that of the fluorescence intensity, suggesting that calcium liberation persists even while the fluorescence begins to decline. Rates of rise of signal mass corresponded to Ca2+ currents of 0.4‐2.5 pA. Measurements of signal mass from different events showed a continuous, exponential distribution, arising through variability in magnitude and duration of calcium flux. We conclude that localized calcium transients in the oocyte represent a continuum of events involving widely varying amounts of calcium liberation, rather than falling into separate populations of ‘fundamental’ and ‘elementary’ events (blips and puffs) involving, respectively, single and multiple InsP3 receptor channels. This variability probably arises through stochastic variation in both the number of channels recruited and the duration of channel opening.

1. The activation of elementary calcium release events ('puffs') and their co-ordination to generate calcium waves was studied in Xenopus oocytes by confocal linescan imaging together with photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. 2. Weak photolysis flashes evoked no responses or isolated calcium puffs, whereas flashes of increasing strength evoked more frequent puffs, often occurring in flurries as abortive waves, and then a near-simultaneous calcium liberation originating at multiple sites. The numbers of sites activated increased initially as about the fourth power of photoreleased [InsP3]. 3. Following repeated, identical photolysis flashes, puffs arose after stochastically varying latencies of a few hundred milliseconds to several seconds. The cumulative number of events initially increased as about the third power of time. No rise in free [Ca2+] was detected preceding the puffs, suggesting that this co-operativity arises through binding of multiple InsP3 molecules, rather than through calcium feedback. 4. The mean latency to onset of calcium liberation shortened as about the square of the flash strength, and the dispersion in latencies between events reduced correspondingly. 5. Weak stimuli often evoked coupled puffs involving adjacent sites, and stronger flashes evoked saltatory calcium waves, propagating with non-constant velocity. During waves, [Ca2+] rose slowly between puff sites, but more abruptly at active sites following an initial diffusive rise in calcium. 6. Initial rates of rise of local [Ca2+] at release sites were similar during puffs and release induced by much (> 10-fold) greater [InsP3]. In contrast, macroscopic calcium measurements averaged over the scan line showed a graded dependence of rate of calcium liberation upon [InsP3], due to recruitment of additional sites and decreasing dispersion in activation latencies. 7. We conclude that the initiation of calcium liberation depends co-operatively upon [InsP3] whereas the subsequent regenerative increase in calcium flux depends upon local calcium feedback and is largely independent of [InsP3]. Wave propagation is consistent with the diffusive spread of calcium evoking regenerative liberation at heterogeneous discrete sites, the sensitivity of which is primed by InsP3.

A large percentage of drugs fail in clinical studies due to cardiac toxicity; thus, development of sensitive in vitro assays that can evaluate potential adverse effects on cardiomyocytes is extremely important for drug development. Human cardiomyocytes derived from stem cell sources offer more clinically relevant cell-based models than those presently available. Human-induced pluripotent stem cell-derived cardiomyocytes are especially attractive because they express ion channels and demonstrate spontaneous mechanical and electrical activity similar to adult cardiomyocytes. Here we demonstrate techniques for measuring the impact of pharmacologic compounds on the beating rate of cardiomyocytes with ImageXpress Micro and FLIPR Tetra systems. The assays employ calcium-sensitive dyes to monitor changes in Ca 2+ fluxes synchronous with cell beating, which allows monitoring of the beat rate, amplitude, and other parameters. We demonstrate here that the system is able to detect concentration-dependent atypical patterns caused by hERG inhibitors and other ion channel blockers. We also show that both positive and negative chronotropic effects on cardiac rate can be observed and IC 50 values determined. This methodology is well suited for safety testing and can be used to estimate efficacy and dosing of drug candidates prior to clinical studies.

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Ca 2+ liberation by inositol 1,4,5-trisphosphate (IP 3 ) is quantal', in that low [IP 3 ] causes only partial Ca 2+ release, but further increasing [IP 3 ] evokes more release. This characteristic allows cells to generate graded Ca 2+ signals, but is unexpected, given the regenerative nature of Ca 2+ -induced Ca 2+ release through IP 3 receptors. Two models have been proposed to resolve this paradox: (i) all-or-none Ca 2+ release from heterogeneous stores that empty at varying [IP 3 ]; and (ii) phasic liberation from homogeneously sensitive stores. To discriminate between these hypotheses, we imaged subcellular Ca 2+ puffs evoked by IP 3 in Xenopus oocytes where release sites were functionally uncoupled using EGTA. Puffs were little changed by 300 mM intracellular EGTA, but sites operated autonomously and did not propagate waves. Photoreleased IP 3 generated¯urries of puffsÐ different to the prolonged Ca 2+ elevation following waves in control cellsÐand individual sites responded repeatedly to successive increments of [IP 3 ]. These data support the second hypothesis while refuting the ®rst, and suggest that local Ca 2+ signals exhibit rapid adaptation, different to the slower inhibition following global Ca 2+ waves.