All three IP(3) receptor subtypes generate Ca(2+) puffs, the universal building blocks of IP(3)-evoked Ca(2+) signals.

All three subtypes of inositol 1,4,5-trisphosphate receptor (IP3R) are intracellular Ca2+ channels that are co-regulated by IP3 and Ca2+ This allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs that reflect the coordinated opening of a few clustered IP3Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca2+ signals in HEK cells expressing all three IP3R subtypes or a single native subtype. Ca2+ puffs are less frequent in cells expressing one IP3R subtype, commensurate with them expressing fewer IP3Rs than wild-type cells. However, all three IP3R subtypes generate broadly similar Ca2+ puffs with similar numbers of IP3Rs contributing to each. This suggests that IP3R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP3R expression levels. The Ca2+ puffs evoked by IP3R2 had slower kinetics and more prolonged durations, which may be due to IP3 binding with greater affinity to IP3R2. We conclude that Ca2+ puffs are the building blocks for the Ca2+ signals evoked by all IP3Rs.

Mataragka S ，Taylor CW 《-》

Dissociation of inositol 1,4,5-trisphosphate from IP(3) receptors contributes to termination of Ca(2+) puffs.

Ca2+ puffs are brief, localized Ca2+ signals evoked by physiological stimuli that arise from the coordinated opening of a few clustered inositol 1,4,5-trisphosphate receptors (IP3Rs). However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.

Smith HA ，Taylor CW 《-》

All three IP(3) receptor isoforms generate Ca(2+) puffs that display similar characteristics.

Inositol 1,4,5-trisphosphate (IP3) evokes Ca2+ release through IP3 receptors (IP3Rs) to generate both local Ca2+ puffs arising from concerted openings of clustered IP3Rs and cell-wide Ca2+ waves. Imaging Ca2+ puffs with single-channel resolution yields information on the localization and properties of native IP3Rs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IP3Rs. Here, we used TIRF and light-sheet microscopy to image Ca2+ puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IP3R type 1, 2, or 3. Photorelease of the IP3 analog i-IP3 in all three cell lines evoked puffs with largely similar mean amplitudes, temporal characteristics, and spatial extents. Moreover, the single-channel Ca2+ flux was similar among isoforms, indicating that clusters of different IP3R isoforms contain comparable numbers of active channels. Our results show that all three IP3R isoforms cluster to generate local Ca2+ puffs and, contrary to findings of divergent properties from in vitro electrophysiological studies, display similar conductances and gating kinetics in intact cells.

Lock JT ，Alzayady KJ ，Yule DI ，Parker I ... -  《-》

Endogenous signalling pathways and caged IP(3) evoke Ca(2+) puffs at the same abundant immobile intracellular sites.

The building blocks of 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 small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs evoked by photolysis of caged IP3 or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. 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 stimulation with carbachol or photolysed 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 and the sites become more likely to fire as the IP3 concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.

Keebler MV ，Taylor CW 《-》

KRAP tethers IP(3) receptors to actin and licenses them to evoke cytosolic Ca(2+) signals.

Regulation of IP3 receptors (IP3Rs) by IP3 and Ca2+ allows regenerative Ca2+ signals, the smallest being Ca2+ puffs, which arise from coordinated openings of a few clustered IP3Rs. Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry.

Thillaiappan NB ，Smith HA ，Atakpa-Adaji P ，Taylor CW ... -  《Nature Communications》