Agonist-Induced Ca2+ Signaling in HEK-293-Derived Cells Expressing a Single IP3 Receptor Isoform

In mammals, three genes encode IP3 receptors (IP3Rs), which are involved in agonist-induced Ca2+ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP3R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP3R1, IP3R2, and IP3R3, respectively. All engineered cells responded to ACh with Ca2+ transients in an “all-or-nothing” manner, suggesting that each IP3R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP3 binding to an IP3R isoform they expressed. Based on a mathematical model of intracellular Ca2+ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca2+ permeability of Ca2+ store and showed that all three IP3R isoforms contributed to Ca2+ leakage from ER. The relative Ca2+ permeabilities of Ca2+ stores in IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were evaluated as 1:1.75:0.45. Using the genetically encoded sensor R-CEPIA1er for monitoring Ca2+ signals in ER, engineered cells were ranged by resting levels of stored Ca2+ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP3R isoforms.


Introduction
Extracellular cues regulate diverse cellular functions by involving a variety of surface receptors and intracellular signaling pathways.The mobilization of intracellular Ca 2+ is central to transduction of many first messengers that act through G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) coupled to the phosphoinositide cascade [1][2][3].GPCRs stimulate the phosphoinositide cascade by involving the β1-4 isoforms of phospholipase C (PLC) in a αGqand/or βγGi-dependent manner, while RTKs employ PLCγ [2][3][4][5][6].Once stimulated, PLC produces two second messengers, IP 3 and diacylglycerol, by hydrolyzing the precursor lipid phosphatidylinositol 4,5-bisphosphate.The primary mode of action of IP 3 is to release Ca 2+ from Ca 2+ store through IP 3 receptors (IP 3 Rs) [1,3].Three genes encode IP 3 R subunits IP 3 R1, IP 3 R2, and IP 3 R3, which form primarily homotetrameric IP 3 -gated Ca 2+ channels in the endoplasmic reticulum (ER).Moreover, evidence exists that different IP 3 R subunits also can form heterotetrameric complexes with specific functional and regulatory properties [3,[7][8][9][10].This and alternative splicing of the IP 3 R genes increase the functional heterogeneity of IP 3 -gated channels, posing an additional complexity to studying physiological functions and regulations of IP 3 Rs in cells [11,12].
Most cell types express two or even all three IP 3 R genes [12], implying that any particular IP 3 R subtype cannot cover the reported diversity of intracellular IP 3 /Ca 2+ signaling [1].The expression pattern of IP 3 Rs is not uniform among different tissues and cell Cells 2024, 13, 562 2 of 21 types, suggesting a specific role for individual IP 3 R isoforms or their combinations in cell physiology [12].For instance, IP 3 R1 predominates in Purkinje neurons, cardiac myocytes rely largely on IP 3 R2, and insulin-secreting β-cells, taste cells, and vomeronasal sensory neurons express primarily IP 3 R3 [13][14][15][16][17].
Since agonist-induced IP 3 /Ca 2+ signaling is pivotal to the physiology of apparently all cell types, IP 3 Rs were subjected to intensive studies at molecular, biophysical, and functional levels [1,3,9,10,18].Reportedly, all IP 3 R isoforms are regulated by cytosolic Ca 2+ in a bimodal manner, suggesting that IP 3 Rs hold two allosteric Ca 2+ -binding sites, both activatory and inhibitory [1,3,19,20].Although the activatory site has been identified, the structure and location of the inhibitory site is still debatable [3].Characteristic of IP 3 R regulation is that IP 3 binding increases affinity of the activatory site, and its occupation by Ca 2+ increases the open probability of the IP 3 -gated channel [19].Owing to this interdependent regulation of IP 3 R gating by the primary co-agonists, Ca 2+ ions released from the ER through IP 3 Rs can facilitate their activity.This positive feedback underlies Ca 2+ -induced Ca 2+ release (CICR), the regenerative process that is ubiquitously involved in the generation of diverse Ca 2+ signals [1,6].Serving as a co-agonist at a relatively low level, cytosolic Ca 2+ suppresses the activity of IP 3 Rs by occupying the inhibitory site at higher concentrations [1,3,19].This multimodal control of IP 3 Rs by IP 3 and Ca 2+ is central to diverse modes of intracellular Ca 2+ signaling [1,20].
Previously, we studied Ca 2+ signaling induced by GPCR agonists, including ATP, UTP, adenosine, ACh, 5-HT, and glutamate, in cells of diverse types [29][30][31][32].The assayed cells universally responded to Ca 2+ -mobilizing agonists in an "all-or-nothing" manner: They either were irresponsive to a particular agonist at subthreshold concentrations or generated quite similar Ca 2+ signals at different agonist doses above the threshold (see Figure 1 below).The body of evidence suggests that being a trigger-like self-driven process, CICR finalized agonist transduction by forming cellular responses of a virtually universal shape, regardless of agonist doses [31].Given that assayed cells express multiple IP 3 R isoforms, it remains unclear whether the "all-or-nothing" responsiveness is mediated by a specific IP 3 R subtype or whether each IP 3 R isoform can endow agonist-induced Ca 2+  signaling in cells with this feature.
As demonstrated in previous studies, a cell line expressing a particular IP 3 R isotype represents a promising cellular model for the systematic assay of gating, regulation, pharmacology, and physiology of IP 3 R isoforms [33][34][35][36][37][38][39].Here, we generated several such cell lines by inactivating two out of three IP 3 R genes in HEK-293 cells using the CRISPR/Cas9 approach.Different aspects of intracellular Ca 2+ signaling in these cells were analyzed with Ca 2+ imaging.It was particularly shown that the engineered cells responded to ACh in the "all-or-nothing" manner and that the ACh sensitivity of a given cellular subclone strongly correlated with EC 50 for IP 3 characteristic of an IP 3 R isoform it expressed.A mathematical model of intracellular Ca 2+ signals induced by thapsigargin, a SERCA inhibitor, was proposed, based on which we developed an approach for estimating relative Ca 2+ permeability of Ca 2+ store.Altogether, our results suggest that the engineered cell lines could provide a relatively simple and effective assay of activity, regulation, and pharmacology of individual IP 3 R isoforms.
To induce transient expression of genetically encoded Ca 2+ sensor R-CEPIA1er with reticular location, cells were transfected with the plasmid vector pCMV R-CEPIA1er kindly provided by Masamitsu Iino (Addgene plasmid # 58216; http://n2t.net/addgene:58216,accessed on 1 October 2019; RRID:Addgene_58216) [40].Before the day of transfection, 3-5 × 10 5 cells were placed in the well of 12-well culture plates.For the transfection of cultured cells, the growth medium was replaced with the transfection mixture, containing 800 µL of the growth medium as well as 200 µL OptiMEM media, 2 µL P3000 Reagent, 2 µL Lipofectamine 3000 (all from Invitrogen, Waltham, MA, USA), and 2 µg pCMV R-CEPIA1er.After 24 h of incubation, the transfection mixture was replaced with the normal culture medium.The transfection was considered effective if at least 30% of the transfected cells exhibited sufficient fluorescence in the red spectral range.Next, the transfected cells were subjected to selection in the presence of antibiotic G418 (700 µg/mL) (Corning) for two weeks.The survivor cells yielded a population, which was maintained in the presence of 300 µg/mL G-418, wherein a nearly 70% cell fraction stably expressed R-CEPIA1er.

Ca 2+ Imaging and IP 3 Uncaging
Isolated cells were plated on a photometric chamber (~150 µL), which contained a disposable coverslip (Menzel-Glaser, Braunschweig, Germany) with an attached ellipsoidal resin wall.The chamber bottom was coated with Cell-Tak (Corning, NY, USA), enabling strong cell adhesion.Attached cells were loaded with Fluo-8 at room temperature (23-25 • C) by adding Fluo-8 AM (3 µM) and Pluronic ® F-127 (0.02%) (both from AAT Bioquest, Pleasanton, CA, USA) to the following bath solution (mM): 140 NaCl, 5 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES-NaOH (pH 7.4), 10 glucose.After 30 min of incubation, the cells were rinsed several times with the bath solution and stored for 40 min to complete dye de-esterification.For IP 3 uncaging, the cells were treated with 3 µM Fluo-8 AM + 4 µM caged-Ins(145)P3/PM (SiChem, Bremen, Germany) + 0.02% Pluronic as described above.When necessary, 2 mM CaCl 2 in the bath were replaced with 0.5 mM EGTA + 0.4 mM CaCl 2 , thus reducing free Ca 2+ to nearly 260 nM at 23 • C, as calculated with the Maxchelator program (http://maxchelator.stanford.edu,accessed on 1 October 2019).All used chemicals were applied with a gravity-driven perfusion system, which enabled the complete replacement of the bath solution in the photometric chamber for nearly 2 s.The used salts and buffers were from Sigma-Aldrich (St. Louis, MO, USA); the ACh and inhibitors were from Tocris Bioscience (Bristol, UK).
Experiments were carried out using an inverted fluorescent microscope Axiovert 135 equipped with an objective Plan NeoFluar 20×/0.75(Zeiss, Oberkochen, Germany) and a digital ECCD camera LucaR (Andor Technology, Belfast, Northern Ireland).Apart from a transparent light illuminator, the microscope was equipped with a handmade system for epi-illumination via an objective.The epi-illumination was performed using a bifurcational glass fiber.One channel transmitted irradiation of computer-controlled LEDs, which provided sequential excitation of Fluo-8 or R-SEPIA1er at 480 ± 5 and 572 ± 17 nm, respectively.Their emission was collected at 535 ± 20 and 630 ± 30 nm, respectively.Serial fluorescent images were captured every second and analyzed using NIS-Elements software (version AR 5.30.01) (Nikon, Tokyo, Japan).Deviations of cytosolic and reticular Ca 2+ in individual cells were quantified by a relative change in intensity of Fluo-8 and R-SEPIA1er fluorescence (∆F/F 0 ), respectively.Another channel was connected to a TECH-351 Advanced pulsed solid laser (680 mW) (Laser-Export, Moscow, Russia).This unit operated in a two-harmonic mode and generated not only 351 nm UV light used for Ca 2+ uncaging but also visible light at 527 nm.The last could penetrate an emission channel through non-ideal optical filters and elicited optical artifacts during uncaging.

Generation of Cell Lines with Inactivated IP 3 R Genes
Two out of three IP 3 R genes in HEK-293 cells were inactivated sequentially using CRISPR/Cas9 technology.Firstly, three monoclonal cell lines were generated, HEK-∆IP3R1, HEK-∆IP3R2, and HEK-∆IP3R3, wherein IP 3 R1-, IP 3 R2-, and IP 3 R3 were disrupted, respectively.Next, by inactivating either of two remaining IP 3 R genes in these lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were obtained with solely IP 3 R1, IP 3 R2, or IP 3 R3, respectively, to be functional.The used methods, protocols, and controls are sufficiently detailed in the Supplementary Materials.

Inactivation of IP 3 R1 Gene
The construct used for IP 3 R1 inactivation was engineered on the basis of the AIO-GFP vector that provided expression of Cas9-D10A nickase fused with the enhanced green fluorescent protein (EGFP) [41].This vector was kindly provided by Steve Jackson (Addgene plasmid # 74119; http://n2t.net/addgene:74119,accessed on 30 March, 2017; RRID: Addgene_74119).The appropriate protospacer locus was identified using IP 3 R1 mRNA sequences (GenBank NM_001099952.4;NM_001168272.2;NM_001378452.1;NM_002222.7).Target-specific sgRNAs were designed and cloned into the AIO-GFP vector as described in the Supplementary Materials.The final cAIO-GFP-sgRNA construct was verified by sequencing.WT-HEK cells were transfected with cAIO-GFP-sgRNA using Lipofectamin 3000 (Invitrogen, Waltham, MA, USA).Seventy-two hours after transfection, EGFP-expressing cells were sorted using a FACSAria SORP sorter (BD Biosciences, NJ, USA) and grown as single cells.Once a particular cell monoclone achieved a monolayer, the cells were collected, their genomic DNA was isolated, and a gene fragment containing the target site was amplified using PCR with specific primers.Each amplicon was subjected to in vitro hydrolysis using commercial nuclease Cas9 and synthesized sgRNA to reveal induced mutations in the IP 3 R1 gene in a source clone.Finally, 4 cell clones (HEK-∆IP3R1) were found to contain necessary biallelic mutations inactivating the IP 3 R1 gene, which also were verified by sequencing.Due to exhibiting the highest fraction (~90%) of cells responsive to ACh with Ca 2+ transients, one clone was chosen for future experimentations.

Inactivation of IP 3 R2 and IP 3 R3 Genes
The IP 3 R2 and IP 3 R3 genes were inactivated using the pGuide-it-tdTomato vector (Takara Bio, San Jose, CA, USA) that encoded nuclease Cas9 fused with the red fluorescent protein tdTomato.The appropriate protospacer locus was identified using the IP 3 R2/IP 3 R3 mRNA sequence (GenBank NM_002223.4/NM_002224.4).Target-specific sgRNA-IP 3 R2/IP 3 R3 was designed and cloned into the pGuide-it-tdTomato vector as described in the Supplementary Materials.After verification by sequencing, the final pGuide-it-tdTomato-sgRNA-IP 3 R2/IP 3 R3 construct was transfected into WT-HEK cells.In seventy-two hours, tdTomato-positive cells were collected using a FACSAria SORP sorter and then grown as single cells.Genomic DNA was isolated from a particular monoclone, and a gene fragment containing a target site was amplified using PCR with specific primers.Each amplicon was subjected to in vitro hydrolysis using commercial nuclease Cas9 and synthesized sgRNA to reveal mutations in the IP 3 R2/IP 3 R3 gene in cells of source clones.Overall, 3 HEK-∆IP3R2 and 4 HEK-∆IP3R3 clones were identified to contain inactivating indels in both alleles of the IP 3 R2 and IP 3 R3 genes, which were verified by sequencing.All these HEK-∆IP3R2/HEK-∆IP3R3 clones were assayed with Ca 2+ imaging, and one exhibiting the highest fraction (~90%) of ACh-responsive cells was taken for future experimentations.

Generation of IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK Lines
Cells expressing the only IP 3 R1 gene were generated by disrupting the IP 3 R2 gene in HEK-∆IP3R3 cells using the strategy described above.In a variety of HEK-∆IP3R3-derived subclones, solely one cell clone, IP3R1-HEK, was eventually identified to contain proper biallelic inactivating mutations in both IP 3 R2 and IP 3 R3 genes.
Cells, wherein solely IP 3 R2 or IP 3 R3 was functional, were generated by inactivating the IP 3 R3 or IP 3 R2 gene in HEK-∆IP3R1 cells, respectively.Single-cell clones of each type, IP3R2-HEK and IP3R3-HEK, were eventually obtained (see Supplementary Materials).

RT-PCR and RT-qPCR
For the expression analysis, total RNA was routinely isolated from a particular cell colony (~10 6 cells), using the Gen Elute Mammalian Total RNA Miniprep Kit (Sigma, St. Louis, MO, USA) according to the manufacturer's protocol.Reverse transcription was performed using SuperScript IV VILO Master Mix (Thermo Fisher Scientific, Waltham, MA, USA).PCR amplification of the target sequences was performed using Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and gene-specific primers (Table S2).The primers were intron-spanning and designed to recognize transcript sequences of all known splice variants, should they be characteristic of an assayed gene.Different RNA transcription levels were quantified with the RT-qPCR approach using a real-time PCR instrument DTlight (DNA Technology, Protvino, Russia) and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA).Amplifications were performed starting with a 3 min template denaturation step at 94 • C, followed by 45 cycles of denaturation at 94 • C for 20 s and combined primer annealing/extension at the gene specific primer temperature for 30 s (Table S2).All samples were amplified in triplicate and the mean was obtained for further calculations.Relative quantification of gene expression was calculated using the 2 -∆Ct method and β-Actin gene as an endogenous reference.

Results
Reportedly, cells of the HEK-293 line endogenously express GPCRs of multiple types, including muscarinic, purinergic, chemokine, lysophospholipid, and protease receptors, many of which are coupled to the phosphoinositide cascade [42].We therefore assayed the responsiveness of basic HEK-293 cells (WT-HEK) and their genetically modified offspring to a variety of GPCR agonists with Ca 2+ imaging.Among agonists capable of mobilizing intracellular Ca 2+ , acetylcholine (ACh) was most effective in that it mobilized cytosolic Ca 2+ in most (80-90%) of the assayed cells.So, cell responsiveness to ACh was predominantly analyzed in the experiments described below.WT-HEK cells that expressed all three IP 3 R isotypes (Figure S6) were subjected to gene editing using the CRISPR/Cas9 technology, and three monoclonal cell lines, each expressing a particular IP 3 R isoform, were generated (see Supplementary Materials).Here, we performed the comparative physiological analysis of Ca 2+ signaling in WT-HEK cells as well as in cells of three lines, called IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, which functionally express solely IP 3 R1, IP 3 R2, and IP 3 R3, respectively.

ACh Responses of WT-HEK Cells
In a particular experiment, 100-120 cells loaded with Fluo-8 were assayed simultaneously using Ca 2+ imaging.When applied shortly, ACh elicited Ca 2+ transients in WT-HEK (Figure 1A) and in cells of the derived lines (Figure 2A).In general, both Ca 2+ release from intracellular Ca 2+ store and Ca 2+ entry mediated by a variety of Ca 2+ -permeable channels contributed to agonist-induced mobilization of cytosolic Ca 2+ .Although to our knowledge, HEK-293 cells do not express nicotinic ACh receptors, receptor-operated channels and store-operated channels (SOCs), which serve in apparently all cell types [6], contributed to the ACh responses.
It turned out that the decrease in bath Ca 2+ from 2 mM to 260 nM affected cellular responses to ACh insignificantly (Figure 1A,B), thus indicating that IP 3 -driven Ca 2+ release was mostly responsible for ACh-induced Ca 2+ signals.The rationale for the abovementioned Ca 2+ protocol was that the complete removal of bath Ca 2+ with EGTA initiated dramatic rundown of cell responsivity during prolonged recordings.On the other hand, cells tolerated the reduction in extracellular Ca 2+ to 260 nM, which proportionally, i.e., by the four orders of magnitude, decreased Ca 2+ influx, in fact nullifying its contribution.In addition, the inhibition of PLC with U73122 (3 µM) rendered cells irresponsive to ACh (Figure 1A).These findings indicate that ACh transduction involved primarily muscarinic receptors that were coupled by the phosphoinositide cascade to IP 3 -driveen Ca 2+ release rather than to Ca 2+ entry.
The previous transcriptome analysis suggests that the M3-receptor was the predominant muscarinic isoform expressed in WT-HEK [42].We confirmed this finding and also evaluated the M3 transcripts to be much more abundant compared to the other M-receptors (M1, M4, and M5) expressed in WT-HEK cells (Figures 1C and S7).Consistently, ACh responses completely disappeared in the presence of 10 nM 4-DAMP (Figure 1C), an antagonist specific to the human M3 and M5 receptors [43].
The peculiar feature of ACh responses was their dose dependence.At concentrations below the threshold of 150-200 nM, ACh insignificantly affected intracellular Ca 2+ in WT-HEK cells but the agonist elicited Ca 2+ transients of similar magnitudes at a variety of higher doses (Figure 1E).This "all-or-nothing" fashion was also characteristic of agonistinduced Ca 2+ responses in cells of several other types [29][30][31][32].Such a step-like dose dependence should be intrinsic to agonist transduction that involves CICR, the mechanism capable of producing a large and global Ca 2+ signal of universal shape, regardless of agonist concentrations [31].The involvement of IP 3 R-mediated CICR in the generation of ACh responses (Figure 1E) was verified by the observation that 0.5 s and 1.5 s UV pulses elicited ACh response-like Ca 2+ transients that were similar kinetically and by magnitude (Figure 1F,G), although the former should have uncaged a nearly three times smaller amount of IP 3 .
capable of producing a large and global Ca 2+ signal of universal shape, regardless of agonist concentrations [31].The involvement of IP3R-mediated CICR in the generation of ACh responses (Figure 1E) was verified by the observation that 0.5 s and 1.5 s UV pulses elicited ACh response-like Ca 2+ transients that were similar kinetically and by magnitude (Figure 1F,G), although the former should have uncaged a nearly three times smaller amount of IP3.

Responses of IP3R1-, IP3R2-, and IP3R3-HEK Cells to ACh
In designated experiments, we assayed IP3R1-HEK-, IP3R2-HEK-, and IP3R3-HEK cells and found that irrespective of the line, most (~90%) of them responded to brief ACh pulses with Ca 2+ transients (Figure 2A).Similar to WT-HEK cells (Figure 1E), cells of each particular line responded to the agonist in an "all-or-nothing" manner (Figure 2A).Being associated with CICR (Figure 1F), the "all-or-nothing" responsivity of IP3R1-HEK-, IP3R2-HEK-, and IP3R3-HEK cells was consistent with the idea that each IP 3 R isoform was capable of mediating CICR [44,45].Given the nearly step-like responsiveness of individual cells (Figures 1E and 2A), their sensitivity to ACh could not be characterized by Ca 2+ response magnitude.As an alternative dose dependence, each assayed population was evaluated by a fraction of cells responsive to the agonist at a particular concentration.After being averaged over all the experiments (n = 7-11) (Figure 2B, symbols), the data were fitted using the nonlinear regression approach and the Hill equation (Figure 2B, continuous lines): where F(C) is the fraction of responsive cells at the given ACh concentration C, F 0 is the maximal fraction of ACh responsive cells, C 0.5 is the EC 50 dose, and n is the Hill coefficient.
Based on this approximation, EC 50 for WT-, IP3R1-, IP3R2-, and IP3R3-HEK cells were estimated as 0.16 ± 0.006, 0.21 ± 0.007, 0.41 ± 0.013, and 1.01 ± 0.05 µM, respectively.Thus, by sensitivity to ACh, the assayed cell lines were ranked as WT-HEK ≈ IP3R2-HEK > IP3R1-HEK > IP3R3-HEK.Consistent with our previous studies of agonist-induced Ca 2+ signaling [30,31], cells of all assayed lines responded to ACh with evident delay relative to the moment of agonist application, depending on the dose of the agonist (Figure 2C).We determined the characteristic time of the response lag (τ d ) as a time interval necessary for a Ca 2+ transient to reach the half magnitude (Figure 2C).The common feature of response lags was that τ d markedly decreased with increasing agonist concentration (Figure 2C,D) in a cell-line-specific manner (Figure 2D).Being similarly sensitive to ACh (Figure 2B), WT-HEK and IP3R2-HEK cells showed quite similar dependencies of response lag on ACh concentration (Figure 2B).In the case of IP3R1-HEK and IP3R3-HEK cells, response lag versus ACh dose was shifted to the right (Figure 2D), consistent with lower sensitivities of both lines to ACh (Figure 2B).
Note that recently, we developed a mathematical model of agonist-induced Ca 2+ signaling, which properly simulated cell responsivity of the "all-or-nothing" type [31].This model predicted that for the phosphoinositide cascade with IP 3 R as the only type, its affinity to IP 3 should be a key factor that determines the lag of Ca 2+ responses.Note that based on their affinity to IP 3 , different IP 3 R isoforms follow the sequence IP 3 R2 > IP 3 R1 > IP 3 R3 [10,[45][46][47].It is therefore likely that only sensitivity of the particular IP 3 R isoform to IP 3 determines the dose-response curve characteristic of the cell line wherein it is expressed (Figure 2B).

Thapsigargin Test of WT-, IP3R1-, IP3R2-, and IP3R3-HEK Cells
Several Ca 2+ -transporting systems are universally involved in Ca 2+ homeostasis in resting and stimulated cells [46,47].To interpret the experiments described below, we employed a simplified model of Ca 2+ homeostasis in assayed cells (Figure 3A).It was suggested that a level of cytosolic Ca 2+ was mainly determined by Ca 2+ fluxes through the plasmalemma and ER membrane, although Ca 2+ -accumulating organelles, such as mitochondria, also could shape Ca 2+ signals.As suggested in Figure 3A, IP 3 Rs, Ca 2+ leak channels, and Ca 2+ -ATPase SERCA were pivotal players in ER, while SOCs, the Na + /Ca 2+ exchanger, and Ca 2+ -ATPase PMCA mediated Ca 2+ fluxes through the plasma membrane.
The inhibition of SERCA with thapsigargin is conventionally used to empty Ca 2+ store through spontaneous Ca 2+ release, thus initiating store-operated Ca 2+ entry (SOCE) in unstimulated cells.We employed the classical thapsigargin test to clarify whether resting activity of IP 3 Rs was a factor of Ca 2+ leakage from ER in assayed cells.In a typical experiment, cells were initially stimulated with 1 µM ACh, and Ca 2+ homeostasis in a given cell was considered sufficiently robust if its Ca 2+ response to the agonist was fast and exceeded 2 in terms of ∆F/F 0 (Figure 3B-E, upper traces).Next, cells were treated with 1 µM thapsigargin with 260 nM Ca 2+ in the bath that nullified a contribution of Ca 2+ entry to intracellular Ca 2+ signals.Under these conditions, thapsigargin-elicited Ca 2+ transients were produced by Ca 2+ leakage from ER, which emptied Ca 2+ store and stimulated activity of SOCs, albeit SOCE was not evident at low bath Ca 2+ .Thapsigargin was applied for 600 s, which was a sufficient interval for intracellular Ca 2+ to return apparently to the initial level.The restoration of bath Ca 2+ to 2 mM initiated significant SOCE associated with a marked Ca 2+ response in the cell cytosol (Figure 3B-E; upper fluorescence trace).To quantify Ca 2+ release and Ca 2+ entry, Ca 2+ traces from individual cells (Figure 3B-E; upper traces) were differentiated, and maximal rates of Ca 2+ release (R r ) and Ca 2+ entry (R e ) were determined as appropriate local maximums in the d(F/F 0 )/dt curves (Figure 3B-E; upward peaks in the bottom traces).
Cells 2024, 13, x FOR PEER REVIEW 10 of 21 plasmalemma and ER membrane, although Ca 2+ -accumulating organelles, such as mitochondria, also could shape Ca 2+ signals.As suggested in Figure 3A, IP3Rs, Ca 2+ leak channels, and Ca 2+ -ATPase SERCA were pivotal players in ER, while SOCs, the Na + /Ca 2+ exchanger, and Ca 2+ -ATPase PMCA mediated Ca 2+ fluxes through the plasma membrane.Based on these data, we generated a number of histograms to characterize distributions of R r and R e among robust cells of different lines.Note that satisfactory fitting for all obtained histograms could not be achieved using a single Gaussian function.Instead, it normally required a combination of two or three Gaussians (Figure 4).This implied that each particular cellular line could include two to three cell subpopulations that differed in Ca 2+ leakage and SOCE.The level of luminal Ca 2+ , activities of Ca 2+ leak channels and Ca 2+ pumps, and coupling of Ca 2+ store to SOCs may have varied from cell to cell.
For SOCE observed after the 600 s depletion of Ca 2+ store (Figure 4, right panels), averaged R r was 0.031 ± 0.009, 0.023 ± 0.007, 0.038 ± 0.012, and 0.018 ± 0.006 (∆F/F 0 )s −1 in WT-, IP3R1-, IP3R2-, and IP3R3-HEK cells, respectively.The histograms generated for R r also demonstrated cell-line specificity (Figure 4, right panels).Although IP3R1-and IP3R3-HEK cells were comparable to the distributions (Figure 4, right IP3R1 and IP3R3 panels) and averaged values of SOCE rates, SOC activity was higher in IP3R2-HEK cells and in WT-HEK cells (Figure 4, right panels of WT and IP3R2).These findings point at the possibility that IP 3 R2 could be functionally coupled to Ca 2+ channels that mediate SOCE in WT-and IP3R2-HEK cells.In contrast, in H4IIE liver cells, which also express all three IP3R isoforms, primarily type 1 and, to a lesser extent, type 3, but not type 2, participated in the activation of a CRAC current associated with SOCE [48].
In resting cells, spontaneous activity of IP 3 Rs could be a factor of Ca 2+ leakage from Ca 2+ store [46].Being least active at rest, IP 3 R3 should have contributed to Ca 2+ leakage to a lesser extent compared to the other IP 3 R subtypes.However, it turned out that just Ca 2+ store in IP3R3-HEK cells was most leaky in terms of the initial rate of thapsigargin-induced Ca 2+ release (Figure 4, left panels).Note, however, that the Ca 2+ release rate depended not only on Ca 2+ permeability of the reticular membrane but also on a level of stored Ca 2+ .Thus, the Ca 2+ release rate could not serve as an independent measure of ER permeability to Ca 2+ .To address this issue, we developed a simplified kinetics model of Ca 2+ signals triggered by thapsigargin at low bath Ca 2+ (Figure S13) and found the Ca 2+ permeability P of the ER membrane to be proportional to the ratio (Equation (S11)): where C is the concentration of cytosolic Ca 2+ , dC dt (0) is the initial rate of a Ca 2+ rise triggered by thapsigargin applied at t = 0, T 0 Cdt is the area under Ca 2+ release curve, and T is the time interval necessary for cytosolic Ca 2+ to return to the initial level (Figure 5A).
To employ this formalism, we suggested that Fluo-8 fluorescence was far below saturation.Indeed, normally, thapsigargin-induced Ca 2+ responses did not exceed 2 (Figure 3A), while the dynamic range of Fluo-8 should have been 20 at least [49].Therefore, the measured parameter F/F 0 was nearly proportional to the concentration of cytosolic Ca 2+ .For each assayed cell, we evaluated both the initial rate of Ca 2+ release (Figure 3B-E, bottom panels) and the area under the Ca 2+ -release trace (Figure 5A, hatched area).) ) where N(r) is the number of cells exhibiting Ca 2+ release/entry rate equal to r, and N i , r i , and σ i are constants.
In WT: left panel,

IP3R1-, IP3R2-, and IP3R3-HEK Cells with the Ca 2+ Sensor R-CEPIA1er
To extend the experimental capability of the engineered lines, R-CEPIA1er, the Ca 2+ sensor with a reticular location [40], was heterologously expressed in IP3R1-, IP3R2-, and IP3R3-HEK cells.Being loaded with Fluo-8, R-CEPIA1er-positive cells allowed for simultaneous monitoring of cytosolic and reticular Ca 2+ (Figure 6).ACh stimulated Ca 2+ ) , where N(r) is the number of cells characterized by the rate/area ratio equal to r, and N i , r i , and σ i are constants.In (B): The appropriate histograms of dC dt (0) and T 0 Cdt obtained for particular IP 3 R isoforms are presented in Figure 5B-D (left and middle panels).As illustrated, thapsigargin induced much more massive Ca 2+ release in IP3R3-HEK cells (Figure 5D, middle panel) compared to IP3R1-HEK and IP3R2-HEK cells (Figure 5B,C, middle panels).The individual ratios (Equation (2)) were calculated, and their distributions among cells of the particular subtype were generated (Figure 5B,C, right panels).For IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells, the averaged ratios (Equation (2)) were 1.42 ± 0.49, 2.49 ± 0.94, and 0.64 ± 0.17, respectively, and the differences between them were statistically significant (p < 0.05, ANOVA test).Based on these values of the ratio (2), relative Ca 2+ permeability of the ER membrane was estimated for IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells as 1:1.75:0.45,respectively.Thus, based on Ca 2+ permeability of the ER, assayed cells were arranged as IP3R2-HEK > IP3R1-HEK > IP3R3-HEK.This order was rather consistent with the values of the steady-state open probabilities of IP 3 R2, IP 3 R1, and IP 3 R3 found to be ~0.3,0.1, and <0.1, respectively, at nearly resting conditions (100 nM Ca 2+ , 1 µM IP 3 ) [19].In agreement with the previous report [39], this conformity suggested that spontaneous activity of IP 3 Rs was an essential factor of Ca 2+ leakage from the ER.Note that the total Ca 2+ release for 600 s (Figure 5B-D, middle panels) indicated that even being least permeable to Ca 2+ (Figure 5B-D, right panels), Ca 2+ store in IP3R3-HEK cells lost a larger number of Ca 2+ ions compared to IP3R2-HEK and IP3R1-HEK cells.It was possible only if a resting level of Ca 2+ in IP 3 -regulated Ca 2+ store in IP3R3-HEK cells was essentially higher than one in IP3R2-HEK and IP3R1-HEK cells, provided that the ER volume was invariable among the cell lines.

IP3R1-, IP3R2-, and IP3R3-HEK Cells with the Ca 2+ Sensor R-CEPIA1er
To extend the experimental capability of the engineered lines, R-CEPIA1er, the Ca 2+ sensor with a reticular location [40], was heterologously expressed in IP3R1-, IP3R2-, and IP3R3-HEK cells.Being loaded with Fluo-8, R-CEPIA1er-positive cells allowed for simultaneous monitoring of cytosolic and reticular Ca 2+ (Figure 6).ACh stimulated Ca 2+ transients in the cell cytosol (Figure 5A-C, upper panels) and a synchronous drop in reticular Ca 2+ , which relaxed close to the resting level despite the agonist still being present in the bath (Figure 6A-C, bottom panels).The Ca 2+ ionophore ionomycin (5 µM) applied at low bath Ca 2+ (260 nM) also triggered cytosolic Ca 2+ signals (Figure 6A-C), presumably by penetrating through plasmalemma and increasing Ca 2+ permeability of the reticular membrane.In this case, the low steady-state level of the ionomycin response was achievable if Ca 2+ fluxes mediated by SERCA and ionomycin were precisely balanced.The relative effects of the agonist and ionophore on reticular Ca 2+ were quantified by the A 1 /A 2 ratio, where magnitudes of Ca 2+ signals elicited by ACh (A 1 ) and ionomycin (A 2 ) were determined as indicated in Figure 6B.As summarized in Figure 6D, ionomycin emptied Ca 2+ store in IP3R1-and IP3R3-HEK cells to a much higher extent than ACh did (Figure 6A,C, bottom panels).In contrast, ACh and ionomycin dropped luminal Ca 2+ in IP3R2-HEK cells to comparable levels (Figure 6B, bottom panels).This phenomenon could be plausibly interpreted based on the recent finding that luminal Ca 2+ inhibited activity of IP 3 Rs and related IP 3 -dependent Ca 2+ signals in the cell cytosol [50].This inhibitory effect was presumably mediated by the Ca 2+ -binding protein annexin A1 (ANXA1).With no ANXA1 bound, IP 3 -gated channels were sufficiently active, while at high luminal Ca 2+ (>100 µM), ANXA1 interacted with IP 3 Rs, promoting their inhibition [50].Given that ANXA1 is expressed in WT-HEK cells (Figure S8), the abovementioned ANXA1-mediated regulation could explain why ACh and ionomycin reduced luminal Ca 2+ to close levels in IP3R2-HEK cells but not in IP3R1-HEK and IP3R3-HEK cells.Indeed, our findings suggest that at rest, ER permeability to Ca 2+ was highest in IP3R2-HEK cells (Figure 5B-D, right panels), implying that a resting level of luminal Ca 2+ should have been lower in these cells compared to IP3R1-HEK and IP3R3-HEK cells, provided that SERCA was similarly active in all cell subgroups.If so, IP 3 Rs in IP3R2-HEK cells operated in a mode characterized by a higher open probability at the same IP 3 level [50], thus mediating higher Ca 2+ release despite lower luminal Ca 2+ .
tive effects of the agonist and ionophore on reticular Ca 2+ were quantified by the A1/A2 ratio, where magnitudes of Ca 2+ signals elicited by ACh (A1) and ionomycin (A2) were determined as indicated in Figure 6B.As summarized in Figure 6D, ionomycin emptied Ca 2+ store in IP3R1-and IP3R3-HEK cells to a much higher extent than ACh did (Figure 6A,C, bottom panels).In contrast, ACh and ionomycin dropped luminal Ca 2+ in IP3R2-HEK cells to comparable levels (Figure 6B, bottom panels).This phenomenon could be plausibly interpreted based on the recent finding that luminal Ca 2+ inhibited activity of IP3Rs and related IP3-dependent Ca 2+ signals in the cell cytosol [50].This inhibitory effect was presumably mediated by the Ca 2+ -binding protein annexin A1 (ANXA1).With no ANXA1 bound, IP3-gated channels were sufficiently active, while at high luminal Ca 2+ (>100 µM), ANXA1 interacted with IP3Rs, promoting their inhibition [50].Given that ANXA1 is expressed in WT-HEK cells (Figure S8), the abovementioned ANXA1-mediated regulation could explain why ACh and ionomycin reduced luminal Ca 2+ to close levels in IP3R2-HEK cells but not in IP3R1-HEK and IP3R3-HEK cells.Indeed, our findings suggest that at rest, ER permeability to Ca 2+ was highest in IP3R2- The asterisk indicate the statistically significant difference (ANOVA test, p < 0.05).There was no statistically significant difference between IP3R1-HEK and IP3R3-HEK cells.

Discussion
The Ca 2+ homeostasis in the ER is governed by a complex protein network, which ensures the steady-state Ca 2+ level in the ER lumina at rest, precise Ca 2+ release upon cell stimulation, and effective refilling of Ca 2+ store [47,[51][52][53].At rest, the level of free Ca 2+ in the ER lumina, which ranges between 300 and 800 µM [51], is determined by balance between passive Ca 2+ leakage and active ER refilling by the SERCA pump [47].The inhibition of SERCA with thapsigargin completely depletes a Ca 2+ pool in the ER within minutes, indicating that Ca 2+ constantly leaks from the ER [54].Several mechanisms have been implicated in mediating Ca 2+ leakage, including proteins from the transmembrane BAX inhibitor motif-containing (TMBIM) family, the antiapoptotic protein BCL-2, and a truncated version of SERCA pump SERCA1T [47,54].The effective feedback mechanism preventing the overload of ER with Ca 2+ involves TMCO1 proteins, which are capable of oligomerizing at high luminal Ca 2+ to form transient Ca 2+ leak channels [55].Evidence exists that in resting cells, spontaneous activity of IP 3 Rs and ryanodine receptors (RyRs) could be responsible for a fraction of Ca 2+ influx from the ER [47,54].For instance, in HEK-293 cells, knockout of all three IP 3 R genes markedly slowed spontaneous Ca 2+ release from the ER [39], indicating that resting activity of IP 3 Rs was a significant factor in Ca 2+ leakage.On the other hand, our observations suggest that RyRs contributed negligibly to Ca 2+ leakage from the ER in HEK-293 cells (Figure S12).
The sustained depletion of luminal Ca 2+ is detrimental to cells, as it causes ER stress, inhibits protein synthesis, and provokes apoptosis [56,57].Cells employ a number of mechanisms to counteract the prolonged depletion of Ca 2+ store, including Ca 2+ -binding proteins buffering luminal Ca 2+ [58], coupling of Ca 2+ store depletion to activated SOCE [59], and active reloading of ER with SERCA [60].The core mechanism of SOCE involves stromal interaction molecules (STIMs), basically STIM1 but also STIM2, which serve as sensors of ER Ca 2+ and SOCE regulators [36].Being initiated by Ca 2+ store depletion, the dissociation of Ca 2+ from STIM1 proteins causes their oligomerization and relocation to the specialized membrane contact sites between the plasma membrane and the ER, called the ER-PM junction.In this junction, the cytosolic domains in STIM1 oligomers have an unfurling and elongated conformation necessary to capture Orai channels located in the plasmalemma and enable their opening [36].
In cells of diverse types, IP 3 Rs represents the main conduit for stimulus-dependent Ca 2+ release [3,9,46], and therefore they should be coupled to SOCs, at least functionally.The functional interaction was indeed demonstrated for IP 3 Rs and TRPC channels involved in SOCE [56][57][58]61,62] as well as between IP 3 Rs and ORAI1 [59,60].Moreover, evidence exists that STIM proteins can directly interact with IP 3 Rs [36,63].The recent findings point out that STIM1 forms a complex predominantly with activated IP 3 R [63].Indeed, being a trigger of STIM1 oligomerization, a transient fall in intraluminal Ca 2+ initiated by IP 3 should be most pronounced just in the close vicinity of open IP 3 R.
The cell lines expressing merely one IP 3 R subtype represent a promising cellular model for the systematic assay of gating, regulation, pharmacology, and physiology of individual IP 3 R isoforms.The first vertebrate cellular model suitable for functional analysis of individual IP 3 R isotypes in the same cellular background was established based on chicken lymphoma-derived DT40 cells, wherein all three IP 3 R genes were disrupted (DT40-TKO cells) [33].The heterologous expression of mammalian IP 3 Rs in DT40-TKO cells provided a deep insight into the functionality of individual IP 3 R isotypes.It was particularly demonstrated that constitutive Ca 2+ release through IP 3 R to mitochondria is required for mitochondrial respiration and maintenance of normal cell bioenergetics [34].It turned out that IP 3 R2 delivered Ca 2+ to mitochondria most effectively, although each IP 3 R isotype can support local contact sites of ER and mitochondria [38].The permeabilized cells expressing a particular IP 3 R subunit were employed to assay Ca 2+ release stimulated by IP 3 and its synthetic analogs.The dose-response curves generated for all IP 3 R subtypes yielded the first structure-activity relationships for the key IP 3 analogues [35].Reportedly, IP 3 -induced Ca 2+ release was effective only if each IP 3 R monomer within the tetramer was occupied by IP 3 [36].
Apart from DT40-TKO cells, IP 3 R-defficient HEK-293 and HeLa cells have also been generated by using CRISPR/Cas9 genome editing [36,37,39].Being employed as a heterologous system for systematic expression and assay of mammalian IP 3 Rs, these model lines facilitated the acquisition of a number of interesting findings.It was particularly reported that upon IP 3 uncaging, all three IP 3 R subtypes were capable of mediating Ca 2+ puffs, relatively small and localized Ca 2+ transients.The pathological mutations associated with dysfunction of IP 3 R1 disrupted IP 3 binding, IP 3 -mediated gating, and its regulation by IP 3 R-modulatory proteins [37].Being significantly reduced in HEK293-TKO cells, Ca 2+ leakage from the ER and its refilling were rescued by overexpression of recombinant IP 3 R1 or IP 3 R3 [39].
In the present work, we developed our own cell lines suitable for the analysis of a role of individual IP 3 R isotypes in agonist-induced Ca 2+ signaling.By inactivating two out of three IP 3 R genes in HEK-293 cells with CRISPS/Cas9 technology and employing cell selection methods, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with IP 3 R1, IP 3 R2, and IP 3 R3 being solely functional, respectively.The functional consequence of this meddling in the natural pattern of IP 3 R expression was evaluated by studying certain aspects of Ca 2+ signaling in these genetically modified cells.
In each line, IP3R1-HEK, IP3R2-HEK, or IP3R3-HEK, cells responded to ACh with Ca 2+ mobilization and exhibited "all-or-nothing" responsivity to the agonist (Figure 2), as was the case with WT-HEK cells (Figure 1D).Given this feature, a dose dependence of cell responses was characterized at the population level by the number of cells responsive to ACh at a particular concentration.Based on the EC 50 doses obtained from these dose-response relationships (Figure 2B), ACh sensitivities of the genetically modified cells ranked as IP3R2-HEK > IP3R1-HEK > IP3R3-HEK.Response lags basically obeyed the same sequence (Figure 2C,D).
It should be noted that genome editing with RNA-guided nucleases, such as Cas9, can entail off-target DNA cleavage [61,62].As an additional control, we compared assayed cells based on the expression of muscarinic receptors and several downstream signaling proteins that could be involved in ACh transduction, including G q -and G i1-3 -proteins, which are known to couple a variety of GPCRs to G-protein-regulated PLCβ1-4 [56].The relative expression levels were assessed using RT-qPCR (see Supplementary Materials).
In mammals, five genes encode muscarinic receptors of the M1-M5 subtypes.We identified M1, M3, M4, and M5 transcripts in WT-HEK cells, while M2 transcripts were undetectable (Figure S7A).Among them, the M3 isotype was dominant at the transcript level (Figure S7B).Consistently, physiological evidence validated the central role of the M3 receptor in mediating ACh-induced Ca 2+ mobilization (Figure 1D).Although expression of the identified M receptors somewhat varied from line to line (Figure S9A), M3 transcript levels were statistically indistinguishable (Figure S9A, M3 panel).It thus appears that distinct sensitivities of IP3R1-, IP3R2-, and IP3R3-HEK cells to ACh (Figure 2B) were determined by a mechanism downstream of the M receptors.
Given that the M3 receptor primarily couples to the G q protein [57], the distinct sensitivities of IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells to ACh may be attributed to lineage-specific expression of G q .It was found that the IP3R1-HEK/IP3R2-HEK and IP3R2-HEK/IP3R3-HEK pairs exhibited statistically indistinguishable levels of G q expression (Figure S9B, G q panel).On the other hand, the level of G q transcripts in IP3R3-HEK cells was ~50% lower than in IP3R1-HEK cells (Figure S9B, G q panel).These data revealed no correlation between a level of G q expression in a particular cell line (Figure S9B, G q panel) and its responsiveness to ACh (Figure 2B).In addition, levels of G i1-3 transcripts in all assayed lines were statistically indistinguishable (Figure S9B, G i panels).Hence, the different sensitivities of IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells to ACh (Figure 2B) did not seem correlated with differences in their expression of G proteins (Figure S9B), which coupled M receptors to PLC.
Although expression of PLCβ1-4 was more scattered among cell populations, statistically significant deviations exhibited solely PLCβ3 transcripts (Figure S6C).Given, however, that compared to PLCβ1 and PLCβ2 the level of PLCβ3 transcripts was lower by a factor of 3-10 (Figure S8C), this PLC isoform was presumably a minor contributor to ACh signaling (Figure 2).In summary, the abovementioned results (Figures S7 and S8) indicate that the IP 3 R gene editing has had insignificant impact on the expression of proteins potentially crucial to ACh transduction.
Previously, we developed a mathematical model of agonist transduction that included the phosphoinositide signaling cascade and IP 3 -driven Ca 2+ release through the only type of IP 3 Rs [31].This model properly simulated the characteristic features of agonist-induced Ca 2+ signaling, such as the "all-or-nothing" responsivity of cells (Figure 2A) and the dose dependence of the response lag (Figure 2D).In line with this model, a threshold agonist concentration in a step-like dose dependence of Ca 2+ responses (Figure 4 in [31]) was determined by both a rate of IP 3 production stimulated by an agonist and the affinity of IP 3 binding to IP 3 Rs.Note that the expression analysis (Figure S9) suggests that the efficacy of the ACh transduction pathway, i.e., muscarinic receptor-G protein-PLC-IP 3 production, was likely similar or nearly identical in IP3R1-, IP3R2-, and IP3R3-HEK cells.On the other hand, based on the revealed levels of IP 3 R transcripts (Figure S10), these cells were ranked as IP3R3-HEK > IP3R2-HEK > IP3R3-HEK, although based on ACh responsivity, they were ordered as IP3R2-HEK > IP3R1-HEK > IP3R3-HEK (Figure 2B).The last sequence aligned well with the affinities of the corresponding IP 3 Rs to IP 3 , which followed the order IP 3 R2 > IP 3 R1 > IP 3 R3 [7,11,[58][59][60].It thus appears that the IP 3 affinity of an IP 3 R isoform operating in a particular cell line was a decisive factor that determined sensitivity of the assayed cells to ACh.

Figure 1 .
Figure 1.Stimulated Ca 2+ signals in WT-HEK cells loaded with Fluo-8 4. (A) Evidence that Ca 2+ release is mainly responsible for ACh-induced Ca 2+ transients.Reduction in bath Ca 2+ from 2 mM to 260 nM weakly affected Ca 2+ responses to 0.75 µM ACh.The PLC inhibitor U73122 (3 µM) suppressed MSC responsivity to ACh.Here and below, the applications of compounds are indicated by the straight-line segments above the experimental trace; the data are presented as ΔF/F0, where ΔF = F − F0, F is the instant intensity of cell fluorescence, and F0 is the intensity of cell fluorescence obtained at the very beginning of a recording and averaged over a 20 s interval.(B) Summary of ACh responses at 2 mM and 260 nM Ca 2+ in the bath.Each response at low extracellular Ca 2+ was normalized to a response at 2 mM Ca 2+ in the bath.The data are presented as a mean ± S.D. (81 cells).(C) Relative levels of muscarinic receptor transcripts in WT-HEK cells (mean ± S.D., n = 3) (see Supplementary Materials for details).(D) ACh responses were reversibly suppressed by the M3/M5 antagonist 4-DAMP at 10 nM.(E) Representative responses of WT-HEK cells (n = 256) to ACh applied at different doses, as indicated.(F,G) IP3 uncaging by 0.5 s and 1.5 s UV flashes elicited similar

Figure 1 .
Figure 1.Stimulated Ca 2+ signals in WT-HEK cells loaded with Fluo-8 4. (A) Evidence that Ca 2+ release is mainly responsible for ACh-induced Ca 2+ transients.Reduction in bath Ca 2+ from 2 mM to 260 nM weakly affected Ca 2+ responses to 0.75 µM ACh.The PLC inhibitor U73122 (3 µM) suppressed MSC responsivity to ACh.Here and below, the applications of compounds are indicated by the straight-line segments above the experimental trace; the data are presented as ∆F/F 0 , where ∆F = F − F 0 , F is the instant intensity of cell fluorescence, and F 0 is the intensity of cell fluorescence obtained at the very beginning of a recording and averaged over a 20 s interval.(B) Summary of ACh responses at 2 mM and 260 nM Ca 2+ in the bath.Each response at low extracellular Ca 2+ was normalized to a response at 2 mM Ca 2+ in the bath.The data are presented as a mean ± S.D. (81 cells).(C) Relative levels of muscarinic receptor transcripts in WT-HEK cells (mean ± S.D., n = 3) (see Supplementary Materials for details).(D) ACh responses were reversibly suppressed by the M3/M5 antagonist 4-DAMP at 10 nM.(E) Representative responses of WT-HEK cells (n = 256) to ACh applied at different doses, as indicated.(F,G) IP 3 uncaging by 0.5 s and 1.5 s UV flashes elicited similar Ca 2+ transients that were reminiscent of Ache responses.In (G), the data are presented as a mean ± S.D (26 cells).

Figure 3 .
Figure 3. Ca 2+ signals associated with SERCA inhibition and Ca 2+ store depletion by thapsigargin.(A) Diagram showing the key contributors to intracellular Ca 2+ signals.(B-E) Upper traces, representative Ca 2+ transients elicited in WT-HEK cells (n = 149) (B), IP3R1-HEK (n = 135) (C), IP3R2-HEK (n = 101) (D), and IP3R3-HEK (n = 117) (E) by 1 µM ACh in control, by 1 µM thapsigargin at 260 nM Ca 2+ in the bath, and by 2 mM bath Ca 2+ after 600 s depletion of Ca 2+ store.The bottom panels show derivatives d F F 0 dt of the upper traces.The values of the first and second peaks were taken as measures for the rates of Ca 2+ release (R r ) and Ca 2+ entry (R e ), respectively.

Figure 6 .
Figure 6.Concurrent monitoring of cytosolic and reticular Ca 2+ in Fluo-8-loaded cells expressing the Ca 2+ sensor R-CEPIA1er.(A-C) Representative Ca 2+ signals in the cytosol (upper panels) and in the ER (bottom panels) of 3 individual cells assayed simultaneously, which belong to the IP3R1-HEK (A) (59 cells), IP3R2-HEK (B) (71 cells), or IP3R3-HEK (C) (63 cells) line.In all cases, cells were sequentially stimulated by ACh at 2 mM Ca 2+ and by 5 µM ionomycin at 260 nM Ca 2+ in the bath.For the particular cell line, the ACh dose was chosen to exceed EC50 (Figure 2B) by a factor of about 5. (D) Ratios of R-CEPIA1er responses on cell stimulation by ACh (A1) and ionomycin (A2), whose magnitudes were determined as shown in (B).The data are presented as a mean ± S.D. (n = 17).The airstrikes indicate the statistically significant difference (ANOVA test, p < 0.05).There was no statistically significant difference between IP3R1-HEK and IP3R3-HEK cells.

Figure 6 .
Figure 6.Concurrent monitoring of cytosolic and reticular Ca 2+ in Fluo-8-loaded cells expressing the Ca 2+ sensor R-CEPIA1er.(A-C) Representative Ca 2+ signals in the cytosol (upper panels) and in the ER (bottom panels) of 3 individual cells assayed simultaneously, which belong to the IP3R1-HEK (A) (59 cells), IP3R2-HEK (B) (71 cells), or IP3R3-HEK (C) (63 cells) line.In all cases, cells were sequentially stimulated by ACh at 2 mM Ca 2+ and by 5 µM ionomycin at 260 nM Ca 2+ in the bath.For the particular cell line, the ACh dose was chosen to exceed EC 50 (Figure 2B) by a factor of about 5. (D) Ratios of R-CEPIA1er responses on cell stimulation by ACh (A 1 ) and ionomycin (A 2 ), whose magnitudes were determined as shown in (B).The data are presented as a mean ± S.D. (n = 17).The asterisk indicate the statistically significant difference (ANOVA test, p < 0.05).There was no statistically significant difference between IP3R1-HEK and IP3R3-HEK cells.