Dissociation of inositol 1,4,5-trisphosphate from IP3 receptors contributes to termination of Ca2+ puffs
2023; Elsevier BV; Volume: 299; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2023.102871
ISSN1083-351X
Autores Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoCa2+ 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. 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. IP3Rs puff along: A SNAPpy dance with IP3 and Ca2+Journal of Biological ChemistryVol. 299Issue 3PreviewConcerted openings of clustered inositol 1,4,5-trisphosphate receptors (IP3Rs) result in short, localized Ca2+ bursts, also called puffs, which are crucial regulators of Ca2+-dependent signaling processes. However, the processes regulating Ca2+ puff amplitude (average ∼0.5 ΔF/F0) and duration (at half-maximal; average ∼25-30 ms) have yet to be elucidated. A recent study in JBC by Smith and Taylor determined that Ca2+ puff amplitude is independent of IP3R cluster density and that the termination of IP3R Ca2+ puff is regulated by IP3 dissociation, illuminating the steps of this regulatory dance. Full-Text PDF Open Access Intracellular Ca2+ signals regulate many cellular processes. Most Ca2+ signals are initiated by inositol 1,4,5-trisphosphate (IP3) produced when cell-surface receptors stimulate phospholipase C. IP3 binds to the four subunits of a tetrameric IP3 receptor (IP3R), priming it to bind Ca2+, and triggering the opening of an intrinsic Ca2+-permeable channel, through which Ca2+ flows rapidly from the lumen of the endoplasmic reticulum to the cytosol (1Foskett J.K. White C. Cheung K.H. Mak D.O. Inositol trisphosphate receptor Ca2+ release channels.Physiol. Rev. 2007; 87: 593-658Crossref PubMed Scopus (936) Google Scholar, 2Alzayady K.J. Wang L. Chandrasekhar R. Wagner L.E. 2nd, Van Petegem F. Yule D.I. Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.Sci. Signal. 2016; 9: ra35Crossref PubMed Scopus (118) Google Scholar, 3Prole D.L. Taylor C.W. Structure and function of IP3 receptors.Cold Spring Harb. Persp. Biol. 2019; 11: a035063Crossref PubMed Scopus (103) Google Scholar). Ca2+ puffs are evoked by low stimulus intensities, similar to those likely to occur under physiological conditions. These Ca2+ puffs are brief, localized increases in cytosolic free Ca2+ concentration ([Ca2+]c) that arise from the coordinated opening of a few channels within a cluster (4Parker I. Smith I.F. Recording single-channel activity of inositol trisphosphate receptors in intact cells with a microscope, not a patch clamp.J. Gen. Physiol. 2010; 136: 119-127Crossref PubMed Scopus (45) Google Scholar, 5Smith I.F. Wiltgen S.M. Shuai J. Parker I. Ca2+ puffs originate from preestablished stable clusters of inositol trisphosphate receptors.Sci. Signal. 2009; 2: ra77Crossref PubMed Scopus (67) Google Scholar, 6Keebler M.V. Taylor C.W. Endogenous signalling pathways and caged-IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites.J. Cell Sci. 2017; 130: 3728-3739PubMed Google Scholar, 7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar). Compelling evidence suggests that the amplitude of a Ca2+ puff reports the number of open IP3Rs. This includes evidence that the amplitudes of steps within the falling phase of a Ca2+ puff match those of the very smallest events ('Ca2+ blips'), which report the opening of a single IP3R (4Parker I. Smith I.F. Recording single-channel activity of inositol trisphosphate receptors in intact cells with a microscope, not a patch clamp.J. Gen. Physiol. 2010; 136: 119-127Crossref PubMed Scopus (45) Google Scholar). The coordinated openings of IP3Rs are thought, at least in part, to arise from costimulation of all IP3Rs by IP3 and Ca2+ (1Foskett J.K. White C. Cheung K.H. Mak D.O. Inositol trisphosphate receptor Ca2+ release channels.Physiol. Rev. 2007; 87: 593-658Crossref PubMed Scopus (936) Google Scholar). An additional, but essential, level of regulation is provided by Kras-induced actin-binding protein (KRAP), which licenses clustered IP3Rs to respond to IP3 (8Thillaiappan N.B. Smith H.A. Atakpa-Adaji P. Taylor C.W. KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals.Nat. Commun. 2021; 12: 4514Crossref PubMed Scopus (20) Google Scholar). Most IP3Rs within a cell are mobile, but Ca2+ puffs arise preferentially from immobile clusters of IP3Rs which are tethered near to endoplasmic reticulum-plasma membrane junctions by KRAP (7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar, 8Thillaiappan N.B. Smith H.A. Atakpa-Adaji P. Taylor C.W. KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals.Nat. Commun. 2021; 12: 4514Crossref PubMed Scopus (20) Google Scholar). These subcellular sites are the same whether Ca2+ puffs are evoked by IP3 produced by endogenous signaling pathways or by photolysis of a caged analog of IP3 (ci-IP3) (6Keebler M.V. Taylor C.W. Endogenous signalling pathways and caged-IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites.J. Cell Sci. 2017; 130: 3728-3739PubMed Google Scholar, 9Lock J.T. Smith I.F. Parker I. Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3.Cell Calcium. 2017; 63: 43-47Crossref PubMed Scopus (21) Google Scholar). Ca2+ puffs may both allow local regulation of Ca2+ sensors and contribute to the genesis of global Ca2+ signals, although the role of Ca2+ puffs in the latter is unresolved (10Lock J.T. Parker I. IP3-mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release.eLife. 2020; 9e55008Crossref Scopus (27) Google Scholar). Ca2+-induced Ca2+-release between IP3Rs is potentially explosive, but the mechanisms that terminate the activity of Ca2+ puffs are incompletely understood. Delayed feedback inhibition of IP3Rs by substantial local increases in [Ca2+]c probably contributes (11Bezprozvanny I. Watras J. Ehrlich B.E. Bell-shaped calcium-response curves for Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum.Nature. 1991; 351: 751-754Crossref PubMed Scopus (1451) Google Scholar, 12Vais H. Foskett J.K. Ullah G. Pearson J.E. Daniel Mak D.O. Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating.J. Gen. Physiol. 2012; 140: 697-716Crossref PubMed Scopus (26) Google Scholar, 13Shuai J. Parker I. Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution.Cell Calcium. 2005; 37: 283-299Crossref PubMed Scopus (86) Google Scholar, 14Bentele K. Falcke M. Quasi-steady approximation for ion channel currents.Biophys. J. 2007; 93: 2597-2608Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), but it may not be the only mechanism. Evidence for coupled closing of IP3Rs during the falling phase of a Ca2+ puff (15Wiltgen S.M. Dickinson G.D. Swaminathan D. Parker I. Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels.Cell Calcium. 2014; 56: 157-168Crossref PubMed Scopus (24) Google Scholar) and conflicting evidence implicating luminal Ca2+ in the regulation of IP3Rs (16Vais H. Wang M. Mallilankaraman K. Payne R. McKennan C. Lock J.T. et al.ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.eLife. 2020; 9e53531Crossref PubMed Scopus (15) Google Scholar, 17Rossi A.M. Riley A.M. Dupont G. Rahman T. Potter B.V.L. Taylor C.W. Quantal Ca2+ release mediated by very few IP3 receptors that rapidly inactivate allows graded responses to IP3.Cell Rep. 2021; 37109932Abstract Full Text Full Text PDF Scopus (6) Google Scholar) suggest additional factors that may contribute to termination of Ca2+ puffs. The effect of cytosolic Ca2+ on IP3Rs is determined by whether they have IP3 bound (1Foskett J.K. White C. Cheung K.H. Mak D.O. Inositol trisphosphate receptor Ca2+ release channels.Physiol. Rev. 2007; 87: 593-658Crossref PubMed Scopus (936) Google Scholar). The simplest scheme suggests that IP3 binding allows Ca2+ to stimulate channel opening, while Ca2+ inhibits IP3Rs without IP3 bound (18Taylor C.W. Tovey S.C. IP3 receptors: toward understanding their activation.Cold Spring Harb. Persp. Biol. 2012; 2: a004010Crossref Scopus (178) Google Scholar, 19Marchant J.S. Taylor C.W. Cooperative activation of IP3 receptors by sequential binding of IP3 and Ca2+ safeguards against spontaneous activity.Curr. Biol. 1997; 7: 510-518Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20Adkins C.E. Taylor C.W. Lateral inhibition of inositol 1,4,5-trisphosphate receptors by cytosolic Ca2+.Curr. Biol. 1999; 9: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This scheme suggests that if local increases in [Ca2+]c contribute to terminating Ca2+ puffs, it may be necessary for IP3 to first dissociate from at least one IP3R subunit; in that case, the rate of IP3 dissociation may be an important determinant of how quickly Ca2+ puffs terminate. Furthermore, we might expect coupled closing and inhibition by local increases in [Ca2+]c to be influenced by the density of IP3Rs within the clusters that evoke Ca2+ puffs. These considerations prompted our analyses of the effects of varying IP3R expression and IP3 affinity on Ca2+ puffs. All three subtypes of IP3R evoke similar Ca2+ puffs (21Mataragka S. Taylor C.W. All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals.J. Cell Sci. 2018; 131: jcs220848Crossref PubMed Scopus (30) Google Scholar, 22Lock J.T. Alzayady K.J. Yule D.I. Parker I. All three IP3 receptor isoforms generate Ca2+ puffs that display similar characteristics.Sci. Signal. 2018; 11eaau0344Crossref PubMed Scopus (41) Google Scholar), but IP3R3 offers advantages for experimental analyses. Plasmids encoding IP3R3 are easy to manipulate, there is an excellent IP3R3-selective antibody, and there are high-resolution structures of IP3R3 in different states (23Paknejad N. Hite R.K. Structural basis for the regulation of inositol trisphosphate receptors by Ca2+ and IP3.Nat. Struct. Mol. Biol. 2018; 25: 660-668Crossref PubMed Scopus (68) Google Scholar, 24Azumaya C.M. Linton E.A. Risener C.J. Nakagawa T. Karakas E. Cryo-EM structure of human type-3 inositol triphosphate receptor reveals the presence of a self-binding peptide that acts as an antagonist.J. Biol. Chem. 2020; 295: 1743-1753Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 25Schmitz E.A. Takahashi H. Karakas E. Structural basis for activation and gating of IP3 receptors.Nat. Commun. 2022; 13: 1408Crossref PubMed Scopus (16) Google Scholar). We therefore chose human IP3R3 for our analyses of Ca2+ puffs. By expressing SNAP-tagged IP3R3 in cells devoid of native IP3R, we were able to visualize both IP3Rs and the Ca2+ signals they evoke. Our results establish that functional interactions between IP3Rs constrain their recruitment during the rising phase of a Ca2+ puff and that IP3 dissociation from the IP3R contributes to termination of each Ca2+ puff. To define the subcellular distribution of IP3R3 while measuring the Ca2+ puffs they evoke, human IP3R3 fused to a fast-labeling SNAP-tag (SNAP-IP3R3, Fig. 1A) was expressed in human embryonic kidney (HEK) cells lacking endogenous IP3Rs (HEK-3KO cells) (2Alzayady K.J. Wang L. Chandrasekhar R. Wagner L.E. 2nd, Van Petegem F. Yule D.I. Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.Sci. Signal. 2016; 9: ra35Crossref PubMed Scopus (118) Google Scholar). We used an N-terminal tag because it does not disrupt IP3R function (7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar) and a SNAP-tag (19.4 kDa; smaller than GFP) that allows versatile covalent labeling with fluorophores (Fig. 1B) (26Liss V. Barlag B. Nietschke M. Hensel M. Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.Sci. Rep. 2015; 517740Crossref Scopus (78) Google Scholar). Our use of HEK-3KO cells (2Alzayady K.J. Wang L. Chandrasekhar R. Wagner L.E. 2nd, Van Petegem F. Yule D.I. Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.Sci. Signal. 2016; 9: ra35Crossref PubMed Scopus (118) Google Scholar) and transient expression of SNAP-IP3R3 under control of an inducible promoter ensured that functional responses were entirely mediated by SNAP-IP3R3 homotetrameric channels expressed at appropriate levels (Fig. 1B). Our optimized methods, which required labeling of cells in suspension before plating for analyses using fluorescence microscopy (Fig. 1, A–D), allowed SNAP-IP3R3 to be identified in cells, most of which expressed IP3R3 at modest levels (Fig. 1E) and with the punctate distribution of IP3R clusters typical of native IP3Rs (Fig. 2Aii and see Fig. 3A) (7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar).Figure 2SNAP-tag labeling reliably reports IP3R distribution. A, TIRF images of HEK-SNAP-IP3R3 cells immunostained with Ab-IP3R3 and labeled with SNAP-Cell 647-SiR (SNAP-647) at high (i) and low (ii) levels of SNAP-IP3R3 expression. Scale bars represent 10 μm, 5 μm in enlargements of boxed areas. B, Manders' split coefficient values for colocalization of Ab-IP3R3 fluorescence with SNAP-647 fluorescence (M1) and vice versa (M2) for cells expressing different amounts of IP3R3 (see panel C). Individual values from 22 cells from two independent analyses (color-coded) and mean ± S.D. C, relationship between whole-cell fluorescence intensities from the entire TIRF footprint for SNAP-647 and Ab-IP3R3 staining. Pearson correlation coefficient r = 0.87, p < 0.0001, n = 34 cells from two independent analyses. D, TIRF images of a HEK-SNAP-IP3R3 cell showing punctate Ab-IP3R3 and SNAP-647 fluorescence. Scale bars represent 10 μm, 5 μm in pseudo-colored enlargements of boxed area. E, fluorescence intensity profiles along the lines (1Foskett J.K. White C. Cheung K.H. Mak D.O. Inositol trisphosphate receptor Ca2+ release channels.Physiol. Rev. 2007; 87: 593-658Crossref PubMed Scopus (936) Google Scholar, 2Alzayady K.J. Wang L. Chandrasekhar R. Wagner L.E. 2nd, Van Petegem F. Yule D.I. Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.Sci. Signal. 2016; 9: ra35Crossref PubMed Scopus (118) Google Scholar, 3Prole D.L. Taylor C.W. Structure and function of IP3 receptors.Cold Spring Harb. Persp. Biol. 2019; 11: a035063Crossref PubMed Scopus (103) Google Scholar, 4Parker I. Smith I.F. Recording single-channel activity of inositol trisphosphate receptors in intact cells with a microscope, not a patch clamp.J. Gen. Physiol. 2010; 136: 119-127Crossref PubMed Scopus (45) Google Scholar) shown in D. F, relationship between fluorescence intensities of individual SNAP-647 and Ab-IP3R3 puncta. 2310 puncta from 10 cells from two independent analyses. Pearson correlation coefficient r = 0.85, p < 0.0001. Ab-IP3R3, IP3R3-selective antibody; HEK, human embryonic kidney; IP3R, inositol 1,4,5-trisphosphate receptor; TIRF, total internal reflection fluorescence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Similar Ca2+ puffs are evoked by endogenous IP3R3 and SNAP-IP3R3. A, typical TIRF image of SNAP-IP3R3 in a live cell selected for analysis because the IP3R expression reveals mobile and immobile puncta typical of native expression (7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar) without the clear delineation of reticular ER that occurs with over-expression. Boxed area (enlarged below) shows the region (19.2 × 19.2 μm) from which Ca2+ puffs were recorded (ROIpuffs). The time-overlay of images captured at 0 s (green) and 60 s (magenta) show immobile SNAP-IP3R3 puncta (white). Scale bars represent 10 μm, 5 μm in enlargements. B, fluorescence traces show Ca2+ puffs evoked by photolysis of ci-IP3 (250-ms UV flash, arrows) in a HEK-IP3R3 or HEK-SNAP-IP3R3 cell. Cal520 fluorescence (F) was recorded in TIRF from a small region (1.76 × 1.76 μm), wherein Ca2+ puffs occurred repeatedly and is expressed relative to fluorescence recorded before the UV flash (F0). Enlargements of boxed areas show individual Ca2+ puffs. C and D, frequency and latency (time from UV flash to first Ca2+ puff) of Ca2+ puffs evoked through SNAP-IP3R3 and endogenous IP3R3. Results show individual cells and mean ± S.D. E, measured properties of Ca2+ puffs (F/F0): amplitude (baseline to peak), rise time (20% to 100% peak amplitude), decay time (100% to 20% peak amplitude), and duration (at half-maximal amplitude). F and G, mean amplitudes (F) and mean kinetic properties (G) of Ca2+ puffs evoked by IP3R3 and SNAP-IP3R3. Results show mean values from single cells and mean ± S.E.M. Results (C, D, F, and G) are from 19 cells from five independent experiments for IP3R3 and from 18 cells from three independent experiments for SNAP-IP3R3. ns p > 0.05, unpaired Student's t test. HEK, human embryonic kidney; IP3R, inositol 1,4,5-trisphosphate receptor; ROIpuffs, ROI from which Ca2+ puffs were recorded; TIRF, total internal reflection fluorescence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We confirmed the specificity of an antibody to IP3R3 (Ab-IP3R3) (27Lagos-Cabre R. Ivanova A. Taylor C.W. Ca2+ release by IP3 receptors is required to orient the mitotic spindle.Cell Rep. 2020; 33108483Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar) in immunocytochemical analyses by demonstrating that Ab-IP3R3 stains puncta in HEK-SNAP-IP3R3 cells, but not in HEK-3KO cells (Fig. S1). HEK-SNAP-IP3R3 cells labeled with SNAP-Cell 647-SiR (hereafter, SNAP-647) were immunostained with Ab-IP3R3 and imaged using total internal reflection fluorescence (TIRF) microscopy. The results demonstrate colocalization of immunostaining and SNAP-647 fluorescence (Fig. 2, A, B, D, and E), a tight linear correlation between the intensities of whole-cell immunostaining and SNAP-647 fluorescence (Fig. 2C), and a linear correlation for individual puncta between Ab-IP3R3 and SNAP-647 fluorescence intensities (Figs. 2F and S2). The results so far (Figs. 1 and 2) demonstrate that transfection of HEK-3KO cells with SNAP-IP3R3 and subsequent labeling with SNAP-647 allows near-native levels of IP3R expression and reliable detection of all IP3R3. We used TIRF microscopy to compare Ca2+ puffs evoked by photolysis of ci-IP3 in HEK-IP3R3 and HEK-SNAP-IP3R3 cells. Since the latter were transiently transfected (Fig. 1B), individual cells differed in their expression of SNAP-IP3R3 (Fig. 1E), we therefore selected cells in which the distribution of IP3R resembled that observed in HeLa cells with tagged endogenous IP3R (7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar), namely cells with comparatively low SNAP-647 fluorescence intensities in which most IP3R puncta were mobile and a smaller fraction were immobile (Fig. 3A). The results demonstrate that Ca2+ puffs occurred with indistinguishable frequencies and after similar latencies in the two cell types (Fig. 3, B–D). Furthermore, the properties of individual Ca2+ puffs (mean amplitudes, rise times, decay times, and durations) were also indistinguishable for HEK-IP3R3 and HEK-SNAP-IP3R3 cells (Figs. 3, E–G and S3). Our results establish that HEK-SNAP-IP3R3 cells after labeling with SNAP-647 allow reliable identification of all IP3Rs within a cell and unperturbed IP3-evoked Ca2+ puffs. We use these cells to explore the effects of varying IP3R expression and the rate of IP3 dissociation from IP3R on the properties of Ca2+ puffs. We used SNAP-647 fluorescence intensity measured from the TIRF footprint of the region of interest (ROI) from which Ca2+ puffs were recorded (ROIpuffs, 19.2 × 19.2 μm) to report SNAP-IP3R3 expression in individual HEK-SNAP-IP3R3 cells and compared it with the properties of the Ca2+ puffs evoked by photolysis of ci-IP3. The results demonstrate that over about a 60-fold range of expression there is a positive correlation between SNAP-IP3R3 expression and the frequency of Ca2+ puffs (Fig. 4A): Ca2+ puffs are more frequent in cells with more IP3Rs. The latency to the first detected Ca2+ puff after the photolysis flash decreased as SNAP-IP3R3 expression increased (Fig. 4B). However, the mean properties of individual Ca2+ puffs (amplitude, rise and decay times, and durations) were similar at all levels of SNAP-IP3R3 expression (Fig. 4, C–F). We next asked how IP3R are distributed within cells expressing different numbers of IP3R. We restricted this analysis to HEK-SNAP-IP3R3 cells in which the SNAP-647 fluorescence intensity measured from ROIpuffs was <20,000 fluorescence units because at higher levels of expression, it was impossible to reliably distinguish individual puncta. The results demonstrate that both the number of SNAP-IP3R3 puncta (Fig. 5A and B) and their mean fluorescence intensity (Fig. 5, A and C) were increased in cells expressing more SNAP-IP3R3. The rightward shifts in the fluorescence intensity distributions for individual puncta as IP3R expression increased indicates that when cells express more IP3Rs, most puncta contain more IP3Rs (Fig. S4). This analysis indicates that clusters of IP3Rs within the TIRF field are more abundant and each cluster includes more IP3Rs in cells with more IP3Rs. These results demonstrate that across a wide range of SNAP-IP3R3 expression (∼60-fold), the properties of individual Ca2+ puffs, including their mean amplitudes, are preserved, but they occur after shorter latencies and they are more frequent in cells with more IP3Rs. Since only a fraction of IP3R puncta are licensed to respond (7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar), we had to consider whether in cells with most IP3Rs, a few puncta with a normal complement of IP3R might lurk beneath the substantial increase in the average number of IP3Rs per punctum and thereby explain the unaltered properties of individual Ca2+ puffs. To address this issue, we recognize that about 30% of IP3R puncta are competent to evoke Ca2+ puffs (Fig. S5) (7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar) and therefore compared the brightest 30% of puncta in cells with the fewest SNAP-IP3R3 to the dimmest puncta in cells with most SNAP-IP3R3. The results demonstrate that in cells with most IP3Rs, the number of puncta with fluorescence intensities comparable to the brightest puncta of cells with fewest IP3Rs is far too low to underlie the observed number of sites at which Ca2+ puffs occur (Fig. 5D). This analysis demonstrates that in cells expressing many IP3Rs, puncta that contain more IP3Rs evoke Ca2+ puffs of unaltered amplitude. We conclude that the amplitude of Ca2+ puffs, indicating the number of open IP3Rs (4Parker I. Smith I.F. Recording single-channel activity of inositol trisphosphate receptors in intact cells with a microscope, not a patch clamp.J. Gen. Physiol. 2010; 136: 119-127Crossref PubMed Scopus (45) Google Scholar, 5Smith I.F. Wiltgen S.M. Shuai J. Parker I. Ca2+ puffs originate from preestablished stable clusters of inositol trisphosphate receptors.Sci. Signal. 2009; 2: ra77Crossref PubMed Scopus (67) Google Scholar, 6Keebler M.V. Taylor C.W. Endogenous signalling pathways and caged-IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites.J. Cell Sci. 2017; 130: 3728-3739PubMed Google Scholar, 7Thillaiappan N.B. Chavda A.P. Tovey S.C. Prole D.L. Taylor C.W. Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.Nat. Commun. 2017; 8: 1505Crossref PubMed Scopus (97) Google Scholar), is similar in cells expressing very different numbers of IP3Rs. Since IP3R clusters are larger in cells with more IP3Rs, functional interactions between IP3Rs must determine the number of IP3Rs that contribute to a Ca2+ puff. This conclusion prompted further analysis of the mechanisms that might terminate the activity of IP3R during a Ca2+ puff. Since the mechanisms that terminate Ca2+ puffs are unresolved (15Wiltgen S.M. Dickinson G.D. Swaminathan D. Parker I. Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels.Cell Calcium. 2014; 56: 157-168Crossref PubMed Scopus (24) Google Scholar, 28Lock J.T. Smith I.F. Parker I. Spatial-temporal patterning of Ca2+ signals by the subcellular distribution of IP3 and IP3 receptors.Sem. Cell Dev. Biol. 2019; 94: 3-10Crossref PubMed Scopus (19) Google Scholar), we asked whether IP3 dissociation from IP3R contributes to termination. A point mutation (R568Q) within the conserved IP3-binding core (IBC) of IP3R1 causes a 9-fold reduction of its affinity for IP3 (29Dellis O. 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