ICC-SM displayed rhythmic firing of Ca2+transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions.Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca Smooth yncytium . The pacyncytium , dissectyncytium , studiesyncytium , studiesyncytium  and simuyncytium . While t2+ action potentials, and excitation-contraction coupling (The anatomy and distribution of ICC varies from place to place throughout the GI tract. Some areas have only an intramuscular type of ICC (ICC-IM) that are closely aligned with and transduce inputs from excitatory and inhibitory enteric motor neurons . Other rcoupling .−1, mean 14.8. min−1)  . Slow waractions . Colonic2+-activated Cl- channels encoded by Ano1 , and the average minimum separation between cell bodies was 49.3 ± 2.8 μm . Most of the ICC-SM network appeared to be adherent to the submucosal layer of the proximal colon. Isolation of the submucosa by sharp dissection showed that few ICC-SM  remained adherent to the muscularis in the uppermost region (1–2 μm) of the proximal colon.Kit immunoreactivity revealed a dense network of Kit-positive cells in the submucosal layer of the proximal colon . The net± 2.8 μm ; n = 6. /copGFP+ mice expressing the copGFP exclusively in ICC were also used to confirm the distribution of ICC-SM. copGFP-positive cells in the submucosal region were present at an average density of 284 ± 27 cells mm–2 and the average minimum separation between cell bodies was 52.7 ± 2.9 μm . ICC-SM were not resolved at the surface of the muscle layer after removing the submucosal layer . Colonic muscles expressing GCaMP6f exclusively in ICC were used to monitor Ca2+ signaling in ICC-SM. GCaMP6f-positive cells in the submucosal region were present at an average density of 291 ± 36 cells mm–2 (n = 10), and the average minimum separation between cell bodies was 50.6 ± 3.8 μm. Representative images of the GCaMP6f-expressing cells in ICC-SM are shown below in the Ca2+-imaging experiments  that allowed simultaneous optical measurements of Ca2+ transients in ICC-SM and SMCs and tissue displacement . The dual-color mouse allowed simultaneous imaging of the two optogenetic Ca2+ sensors with different fluorescence characteristics . The sensors were expressed in a cell-specific manner in ICC and SMCs to characterize the coordination of signaling between ICC and SMCs . Ca2+ transients recordings were obtained from ICC-SM and SMC at the exact coordinates in the pixel matched images . Muscle displacement, an indicator of muscle contraction, was also measured and displayed latencies of 120 ± 17 ms between the rise of Ca2+ in SMCs and resolvable displacement  and 180 ± 15 ms between activation of Ca2+ transients in ICC-SM and contractile displacement . In a few instances, the time delay between ICC-SM and SMCs was not readily resolved. This could have resulted from a shift in the site of dominant pacemaker activity to a region outside the field of view (FOV). There was close correlation between the temporal latencies of ICC-SM and SMCs to tissue displacement (2 = 0.82), indicating the pacemaker nature of ICC-SM, the coupling between ICC-SM and SMCs and the management of Ca2+ signaling and contractions in SMCs by the pacemaker activity of ICC-SM.We developed a new mouse strain . Similar behavior occurred at similar frequencies  in ICC-SM attached to muscle tissues. Ca2+ waves propagated through the isolated ICC-SM networks with velocities of 219 ± 19 mm/s. Ca2+ transients imaged at ×10 appeared to be global and had average durations of 2.1 ± 0.4 s  and amplitudes of 1.2 ± 0.3 ΔF/F0 . The spatial spread of Ca2+ transients was 36.8 ± 0.4 µm . The average area occupied by Ca2+ transients in the spatiotemporal maps was 51.6 ± 1.6 µm*s .Movement generated by muscle contractions is always an issue when imaging cells in muscle preparations in situ. Therefore, we used preparations of ICC-SM adherent to submucosa that were separated from muscle strips in most experiments. At low-magnification (×10), rhythmic Cale min−1  and aver2+ transients in ICC‐SM is shown in 2+ transients appeared to be sequential in nature , as there was a strong correlation between the occurrence of global Ca2+ transients in multiple ICC-SM cells . Overlays of STMaps of Ca2+ activity in adjacent cells running parallel to each other  . These results suggest that Ca2+ firing in ICC‐SM networks are entrained and propagate cell‐to‐cell.Cell-to-cell propagation of Cach other  showed t period) . Compari2+, however, imaging of ICC-SM at higher power (×60–×100) allowed a more detailed visualization of the subcellular nature of the Ca2+ transients and revealed complex firing patterns. Imaging of ICC-SM networks with high spatial resolution showed that subcellular Ca2+ transients originated from distinct firing sites (n = 25). STMaps constructed from Ca2+ transients in single ICC-SM during 30 s of imaging .Low-power imaging suggested global changes in cell Cang sites ; n = 25. imaging  identifi2+ waves . The num2+ waves ; n = 25.2+ particle analysis  and particle count/frame averaged 0.27 ± 0.1 .We employed Caanalysis  to identeristics . The firnce maps . Occurreng sites . Ca2+ trnetworks . Also aphan once . From pa 0.4 µm2 ; n = 25 27 ± 0.1 ; n = 25.2+-firing sites were most active during the first ~256 ms of a Ca2+ wave, and activity decayed with time. This point was further illustrated by distribution plots showing the average percentage of firing sites discharging at various times during Ca2+ waves . Initially high firing and decay as a function of time suggest that Ca2+ entry mechanisms may be important for: (i) initiating Ca2+ transients and (ii) organizing the occurrence of Ca2+ transients into clusters. It is also possible that Ca2+ stores, loading during the diastolic period between Ca2+ waves, are more excitable at the onset of each Ca2+ wave.Particle analysis also showed that Ca2+ waves ; n = 25.2+ ([Ca2+]o) on Ca2+ transients was investigated to evaluate the importance of Ca2+ influx for the patterning of Ca2+ signaling in ICC-SM. Removal of [Ca2+]o abolished Ca2+ transients in ICC-SM within 10 min . Stepwise reduction in [Ca2+]o (from 2.5 mM to 0 mM) showed that Ca2+ transients decreased in a concentration-dependent manner . ICC-SM Ca2+ PTCL area and count of firing sites were reduced in response to lowering [Ca2+] o . Reducing [Ca2+]o from 2.5 mM (control conditions) to 2 mM caused a reduction in the average PTCL area by 19.6 ± 4.5%  and PTCL count by 25.4 ± 2.9%  with no significant change in the duration of PTCLs . Further reduction of [Ca2+] o to 1 mM reduced Ca2+ transient parameters by 47.2 ± 3.5% PTCL area  and 45.1 ± 4.4% PTCL count  and showed a significant change in the duration of PTCLs . Lowering [Ca2+] o to 0.5 mM also reduced Ca2+ PTCL area by 73.9 ± 3%  and PTCL count by 73.2 ± 2%  with no significant change in the duration of PTCLs . Removal of Ca2+ from the extracellular solution (Ca2+‐free KRB solution containing 0.5 mM EGTA) abolished Ca2+ signals 8–10 min after solution replacement. The Ca2+ transient PTCL area was reduced by 99.4 ± 0.6%  and PTCL count by 99.3 ± 0.7%  with no significant change in the duration of PTCLs . We also noted that the highly organized CTCs occurring in the presence of 2.5 mM [Ca2+] o became less organized as [Ca2+] o was reduced  and evaluated the expression of voltage-dependent and voltage-independent Ca2+ channels by qPCR . The expression levels of Myh11 an SMC marker and Uch11 a pan neuronal marker encoding PGP9.5 were minimal , confirming the purity of ICC-SM sorted by FACS.The apparent dependence on the Can ICC-SM . After i by qPCR . First,  by qPCR . Kit rec2+ channels encoded by Cacna1c and Cacna1d abundantly . Cacna1c showed a 0.012 ± 0.0005 and Cacna1d showed 0.066 ± 0.008 relative to Gapdh . ICC-SM also expressed Cacna1h (CaV 3.2) and to a lesser extent Cacna1g (CaV 3.1), both T-type voltage-dependent Ca2+ channels . Cacna1h expression was abundant in ICC-SM (0.07 ± 0.003 relative to Gapdh). Cacna1g expression was less than Cacna1h 0.0014 ± 0.0001 relative to Gapdh .ICC-SM expressed L-type voltage-dependent Cato Gapdh ; n = 4. channels ; n = 4. to Gapdh ; n = 4.2+ stores may also be important for mediation and shaping of Ca2+ signals in ICC-SM. Store‐operated Ca2+ entry (SOCE) has been established as a mechanism for filling stores upon depletion . Orai1 expression was 0.02 ± 0.001 relative to Gapdh and Orai2 was 0.074 ± 0.008 relative to Gapdh .Maintenance and refilling of cellular Caepletion . Contribs of ICC . Colonicresolved ; n = 4. to Gapdh ; n = 4.2+ channels  were expressed in ICC-SM, and Ca2+ transients showed dependence on the Ca2+ gradient. Previous studies have reported that L-type channel antagonists inhibit slow waves in the colon (2+ entry via L-type channels to Ca2+ transients in ICC-SM. Nicardipine (1 μM) abolished Ca2+ transients in ICC-SM . Firing site occurrence maps  describe the inhibitory effects of nicardipine on Ca2+ transients. Ca2+ PTCL area was reduced to 10.5 ± 4.7%  and PTCL count was reduced to 12.3 ± 4.8% . The number of firing sites also decreased to 8.4 ± 3% in the presence of nicardipine . Isradipine inhibits CaV 1.2 and CaV 1.3 equally . ICC-SM Ca2+-firing was reduced in the presence of isradipine as shown in the firing sites occurrence maps , and PTCL count was reduced to 19.5 ± 6% . The number of firing sites was inhibited by isradipine to 21.5 ± 6% . These data show that ICC-SM Ca2+ transients depend upon Ca2+ influx via L-type Ca2+ channels, and since isradipine had nearly the same or a lesser effect on Ca2+ transients than nicardipine, CaV 1.2 appear dominant as the Ca2+ entry mechanism.L-type Cahe colon . Therefon ICC-SM ; n = 8. nce maps ; n = 8 d5 ± 4.7% ; n = 8 a3 ± 4.8% ; n = 8. ardipine ; n = 8.  equally . Isradipn ICC-SM ; n = 7. nce maps . PTCL arnce maps ; n = 7, 9.5 ± 6% ; n = 7. 1.5 ± 6% ; n = 7. 2+ channels have been identified as the dominant voltage-dependent Ca2+ conductance responsible for Ca2+ entry and propagation of slow waves in ICC-MY of the small intestine (2+ and mibefradil) reduced the rate-of-rise of the upstroke component of the slow waves and higher concentrations attenuated slow wave activity , TTA-A2 (10 μM) and Z-944 (1 μM). NNC 55–0396 reduced Ca2+ transient firing  and PTCL count was reduced to 35.6 ± 4.5% . The number of firing sites was reduced by NNC 55–0396 to 37.6 ± 4.3% . The duration of PTCLs was also reduced by NNC 55–0396 to 73 ± 5.2% . TTA-A2 showed similar inhibitory effects on ICC-SM Ca2+ transients  and PTCL count was reduced to 44.4 ± 6.2%  The number of firing sites was also reduced by TTA-A2 to 43.4 ± 6% . Z-944 significantly reduced ICC-SM Ca2+ transients but was somewhat less effective than NNC 55–0396 or TTA-A2. PTCL area was reduced to 56 ± 10% , and PTCL count was reduced to 53 ± 12%  The number of firing sites was also reduced by Z-944 to 53.4 ± 11% . The data suggest that T-type Ca2+ channels also contribute to the initiation and organization of Ca2+ transients in ICC-SM.T-type Cantestine ; . T-typactivity . As repot firing , Ca2+ trnce maps  and firit firing . PTCL art firing ; n = 9 a6 ± 4.5% ; n = 9. 6 ± 4.3% ; n = 9. 3 ± 5.2% ; n = 9. ansients . PTCL aransients ; n = 7 a4 ± 6.2% ; n = 7 T3.4 ± 6% ; n = 7. 56 ± 10% ; n = 5, 53 ± 12% ; n = 5 T.4 ± 11% ; n = 5. 2+ transients in ICC-SM depend on voltage-dependent Ca2+ influx mechanisms (V 1.3 and CaV 1.2 and CaV 3.2) was further examined under conditions of membrane hyperpolarization induced by activation of KATP channels. KATP is functional in colonic SMCs but not in ICC  and PTCL count was increased to 121.8 ± 17.8% . The number of firing sites was not affected by pinacidil . The effects of pinacidil on global Ca2+ transients were evaluated. Pinacidil significantly reduced the duration of global Ca2+ transients from 1.78 ± 0.2 s to 0.89 ± 0.1 s . Reduction in the duration of Ca2+ transients was associated with a tendency for an increase in frequency, but this parameter did not reach significance. Ca2+ oscillation frequency under control conditions was 15.1 ± 1.1 cpm and in the presence of pinacidil was 16.5 ± 1.2 cpm .Experiments described above showed that Cachanisms –10. The t in ICC . Therefochannels  and the y ~10 mV . Pinacid of CTCs . Ca2+ PT ± 0.1 s ; n = 6. 2+ transients in ICC-SM . PTCL area was reduced to 38.5 ± 7.6%  and PTCL count was reduced to 42.3 ± 9.1% . In some regards, these results were surprising as membrane potential hyperpolarization might reduce contributions from L-type Ca2+ channels (CaV 1.2). One explanation is that CaV 1.3, which are abundant in ICC-SM and activate at more negative potentials than CaV 1.2  inhibited Ca2+ transients in ICC-SM to a greater extent. PTCL area was reduced to 9.0 ± 2% , and PTCL count was reduced to 11.2 ± 1.9% . The utilization of two Ca2+ conductances with different ranges of voltage-dependent activation for the initiation of CTCs provides a safety factor that insures persistence of pacemaker activity over a broad range of membrane potentials.The effects of nicardipine were tested in the presence of pinacidil. Under these conditions, nicardipine significantly reduced Can ICC-SM ; n = 5. 5 ± 7.6% ; n = 5 a3 ± 9.1% ; n = 5.  CaV 1.2  may cont9.0 ± 2% ; n = 5, 2 ± 1.9% ; n = 5. 2+ channels in the presence of pinacidil unmasked underlying Ca2+ transients that occurred more randomly than the clustered transients occurring normally . Underlying Ca2+ events were more frequent in the presence of pinacidil and nicardipine and increased again upon addition of NNC 55–0396 .Inhibiting voltage-dependent Canormally . We tabu 55–0396 ; n = 5.2+ signaling in ICC-MY in the small intestine depends not only on Ca2+ influx but also on Ca2+ release from intracellular stores  reduced, but did not block, Ca2+ transient firing in ICC-SM  and PTCL count was reduced to 22 ± 10% . The number of firing sites was reduced by thapsigargin to 21 ± 11% . The duration of PTCLs was increased by thapsigargin to 126.7 ± 9% . Cyclopiazonic acid , another SERCA antagonist, also reduced Ca2+ transient firing  and PTCL count was reduced to 29.1 ± 6.3% . The number of firing sites was reduced by CPA to 56 ± 9.5% . There was no significant change in the duration of PTCLs in the presence of CPA .Previous studies have demonstrated that Car stores . Ca2+ reow waves . The roln ICC-SM . PTCL arn ICC-SM ; n = 6 a22 ± 10% ; n = 6. 21 ± 11% ; n = 6. 6.7 ± 9% ; n = 6. t firing . PTCL art firing ; n = 5 a1 ± 6.3% ; n = 5. 6 ± 9.5% ; n = 5. e of CPA ; n = 5.3Rs amplify and sustain Ca2+ signaling via direct localized release of Ca2+ or Ca2+-induced Ca2+ release (CICR) (3Rs to Ca2+ transients in ICC-SM were therefore investigated. Ryanodine (100 μM) significantly reduced Ca2+ event firing in ICC-SM  and PTCL count was reduced to 80.9 ± 2.7% . The number of firing sites was also reduced by ryanodine to 75.7 ± 2.1% . There was no significant change in the duration of PTCLs in the presence of ryanodine . We also noted that the greatest effects of ryanodine on Ca2+ transients occurred after the first ~300 ms of CTCs . Thus, ryanodine shortens the durations of the CTCs. Ca2+ release mechanisms via RyRs contribute to the overall patterns of Ca2+ waves in ICC-SM, as shown by the distribution plots of average percentages of firing sites during a Ca2+ wave .ER release channels RyRs and IPe (CICR) . Contribn ICC-SM . PTCL arn ICC-SM ; n = 4 a9 ± 2.7% ; n = 4. 7 ± 2.1% ; n = 4. yanodine ; n = 5.  of CTCs ; n = 4. a2+ wave ; n = 4.3R antagonist) also reduced Ca2+ events in ICC-SM . PTCL area was significantly reduced to 78 ± 5.8%  and although both PTCL count and number were reduced to 75.3 ± 10.5% and 74.8 ± 9.3%, respectively, these effects did not reach statistical significance . Xestospongin C displayed inhibitory effects similar to ryanodine; most of the inhibition of Ca2+ events occurred after the first ~400 ms of CTCs . Thus, Ca2+ release via IP3Rs also contributes to the overall pattern of Ca2+ waves in ICC-SM, as shown by the distribution plots of average percentages of firing sites during a Ca2+ wave .Xestospongin C  as secondary tests of the contributions of IP3Rs and RyRs in CTCs. 2-APB reduced Ca2+ PTCL area to 61.1 ± 14.5%  and reduced PTCL count to 58 ± 13.2% . The number of firing sites was also reduced by 2-APB to 63.7 ± 11.3% . Tetracaine reduced Ca2+ PTCL area to 85 ± 5.1%  and reduced PTCL count to 69.5 ± 7.8% . The number of firing sites was also reduced by tetracaine to 71.3 ± 5.9% .2-APB (100 μM) and tetracaine were also tested 100 μM; ; n = 5 a2+ entry channels . Previous studies have shown SOCE to be important for maintenance of Ca2+ stores and sustaining Ca2+ release from the ER  reduced the firing frequency of CTCs , and PTCL count was reduced to 48 ± 7% . The number of firing sites was also inhibited by GSK 7975A to 47.5 ± 4.1% . There was no significant change in the duration of PTCLs in the presence of GSK 7975A .The effects of 2-APB could be non-specific and may include effects on store-operated Cam the ER . ICC-SM  of CTCs . Firing  of CTCs  and PTCL of CTCs . Ca2+ PT4 ± 9.4% ; n = 7,  48 ± 7% ; n = 7. 5 ± 4.1% ; n = 7. SK 7975A ; n = 7.2+ transients responsible for the pacemaker function of ICC-SM that contributes to contractile patterns in colonic motility. The sequence of activation from ICC-SM to colonic SMCs was quantified using two optogenetic sensors, expressed specifically in ICC or SMCs. Correlation analysis demonstrated a 1:1 relationship between CTCs in ICC-SM and Ca2+ signaling and contractile responses in SMCs. The CTCs in ICC-SM consisted of ~2 s bursts of activity from multiple sites within cells. Organization of the Ca2+ transients into clusters was due to voltage-dependent Ca2+ entry that appeared to be due to activation of both high-voltage activated Ca2+ channels (L-type encoded by Cacna1c and Cacna1d in ICC-SM) and low-voltage activated Ca2+ channels (T-type encoded by Cacna1h and possibly Cacna1g in ICC-SM). A portion of the Ca2+ transients making up CTCs were due to Ca2+ entry, as the earliest Ca2+ transients resolved in a cluster were not blocked by thapsigargin, CPA or antagonists of ryanodine and IP3 receptors. The earliest events were blocked by nicardipine, suggesting that L-type Ca2+ channels are the dominant Ca2+ entry pathway at basal resting potentials. Ca2+ transients in ICC-SM were not as sensitive to block by STIM and Orai, as in other ICC . Correlation analysis demonstrated the coherence of these events. Ca2+ transients, lasting for about 2 s, propagated without decrement through networks of ICC-SM and preceded and likely initiated Ca2+ signaling and contractions in SMC, as was also suggested by intracellular microelectrode recordings from cells along the innermost surface of canine colonic muscles  and red (RCaMP1.07) Ca muscles . ICC-SM 2+ imaging in most smooth muscle tissues.Slow waves with characteristics similar to those found in the stomach and small intestine (i.e. relatively fast upstroke depolarization and a plateau phase) are generated along the submucosal surface of the CM layer in the colon . It was 2+ transients in low resolution imaging partitioned into clusters of localized Ca2+ transients when viewed with a ×60 objective at 30 frames/s or at higher acquisition speeds. Summation of the clustered events reproduced the frequency and duration of the Ca2+ waves observed at low resolution. Multiple firing sites, averaging ~8 per cell, were identified. This pattern of clustered Ca2+ transients (CTCs) was also observed in ICC-MY of the small intestine, the pacemaker cells in that region  activate at relatively hyperpolarized membrane potentials and their currents are partially inhibited by dihydropyridines (~50–70% of current density block) in comparison to Cacna1c gene products (CaVα1C)  , but isr equally . The fac in mice ; however2+ conductances with different voltage-dependent properties coordinate clustering of Ca2+ transients provides a safety factor for preservation of pacemaker activity over a broad range of membrane potentials. In spite of overarching changes in membrane potential that might influence the availability of ion channels with narrow voltage-ranges, the broader range of activation potentials offered by expression and function of both L-type and T-type Ca2+ channels might protect against voltage-dependent inhibition of pacemaker activity. L-type channels are activated at less polarized potentials than T-type channels (2+ entry mechanism from L-type to T-type Ca2+ channels. This concept was demonstrated by the decreased inhibitory effects of nicardipine and increased effects of NNC 55–0396 on Ca2+ transients after exposure of tissues to pinacidil. The opposite might be true if the SIP syncytium experiences a depolarizing influence (e.g. neurogenic or humerogenic).Having three independent voltage-dependent Cachannels . Thus, achannels  may tendATP channels in SMCs (V/dtmax of the upstroke depolarization and decreases the duration of slow waves (Vα1D and T-type channels (CaVα1H) at more hyperpolarized potentials. The decrease in the duration of CTCs may be due to a relative shift in the importance of T-type vs. L-type Ca2+ channels with hyperpolarization. In the presence of pinacidil, NNC 55–0396 had increased antagonistic effects on CTCs. Ca2+ currents via T-type channels inactivate rapidly, whereas L-type channel inactivation is slower and incomplete , and frequency of CTCs was greatly reduced at concentrations lower than 1 mM. Our concept is that Ca2+ entry couples to CICR in ICC. Reducing the driving force for Ca2+ entry would be expected to reduce the probability for effective coupling to CICR. CICR would be negligible when Ca2+ entry falls below threshold levels. Concentrations of thapsigargin and CPA that blocked Ca2+ transients quantitatively in other ICC  (The importance of Cather ICC  caused ahannels) .2+ release events, occurring in all ICC  due to activation of ANO1 channels result in spontaneous transient membrane depolarizations (STDs). Temporal summation of STDs is likely to be the generator potentials that activate T-type or L-type Ca2+ currents and initiate propagating slow wave events. In this concept it is logical to suggest that inhibition of Ca2+ release should reduce the duration of the CTCs, and inhibition of Ca2+ entry should inhibit the organizing influence of Ca2+ entry and block CTCs. When CTCs are blocked, stochastic Ca2+ transients may be unleashed, as occur in ICC-IM  in maintaining Ca2+ release events in ICC  Propagation of activity within the ICC-SM network depends upon voltage-dependent Ca2+ entry, and the functions and voltage-dependent properties of three types of Ca2+ conductances appear to provide a safety factor that tends to preserve pacemaker activity over a broad range of membrane potentials  Ca2+ entry also appears to contribute to refilling of stores, as pacemaker activity was not as immediately dependent upon SOCE as in other ICC , possibly through activation of ANO1 channels and depolarization, may underlie the pacemaker functions of ICC-SM. Additional studies will be necessary to resolve these hypotheses in finer detail. The preparation of excised submucosal tissue with adherent ICC-SM removes movement artifacts from imaging and is likely to provide a powerful tool for improving resolution of pacemaker mechanisms and determining how regulatory and pathophysiological factors affect basic pacemaker mechanisms.In summary ICC-SM, as suggested from dissection and electrophysiological experiments , are pacractions , summatemotility . Data frials see . (ii) Cather ICC . (iv) Th+/copGFP mice  were bred in house (/Cre-ERT2+) were gifted from Dr. Dieter Saur .Kitin house . GCaMP6fActa2-RCaMP1.07 mice (tg(RP23-370F21-RCaMP1.07)B3-3Mik/J) express the fluorescent Ca2+ indicator RCaMP1.07 in SMCs under the control of the Acta2 locus promoter/enhancer regions were obtained from Jackson Laboratories . To generate cell-specific expression in two distinct cell types (ICC and SMCs) Acta2-RCaMP1.07 mice were bred with Cre‐ERT2/GCaMP6ffl/flKit mice. The offspring Kit-iCre-GCaMP6f/Acta2-RCaMP1.07 mice were identified by genotyping after receiving tamoxifen which served to delete the STOP cassette in the Cre-expressing cells; resulting in the expression of the fluorescent Ca2+ indicator protein, GCaMP6f. These mice allowed simultaneous, dual color imaging of ICC and SMCs. iCre mice were injected with tamoxifen  at 6–8 weeks of age (2 mg of TAM for three consecutive days), as described previously .tunica muscularis. (2) The submucosa layer was left attached to the tunica muscularis after removal of the mucosa. The isolated submucosal layer preparation provided better imaging of ICC-SM by eliminating motion artifacts associated with muscle contractions. We used the isolated submucosal layer preparation in most cases in this study with the exception of two experiments where muscle attachments were necessary to test important questions (see Results).Colonic segments  were removed from mice after an abdominal incision and placed in Krebs-Ringer bicarbonate solution (KRB). The tissues were cut along the mesenteric border and intraluminal contents were washed away with KRB. Tissues were prepared by blunt dissection in two ways: (1) The submucosa layer was isolated after carefully removing the mucosal layer and the tunica muscularis were fixed in 4% paraformaldehyde and visualized as described previously (http://rsbweb.nih.gov/ij). ICC (copKit) images were visualized using a spinning-disk confocal system .Colonic tissues from wild-type mice were processed to assess distribution of c-Kit immunoreactivity. Whole mounts of submucosal layer after removing the mucosa and eviously . Briefly/copGFP+ mice  were used for evaluations of gene expression in ICC-SM. Cell-specific expression of the fluorescent reporter allows unequivocal identification of ICC  mounted on an upright Nikon Eclipse FN1 microscope equipped with several water immersion Nikon CFI Fluor lenses  . The system is equipped with two solid-state laser lines of 488 nm and 561 nm. The laser lines are combined with a borealis system  to increase laser intensity and uniformity throughout the imaging FOV. The system also has two high-speed electron multiplying charged coupled devices (EMCCD) cameras  to allow dual-color imaging simultaneously and maintain sensitive and fast speed acquisition at full frame of 512 × 512 active pixels as previously described  custom build software ; (2) Fiji/Image J ; (3) Automated Spatio Temporal Map analysis plugin (STMapAuto), https://github.com/gdelvalle99/STMapAuto as described previously (2+ transients (stacks of TIFF images) were imported into Volumetry G8d and motion stabilized, background subtracted and smoothed . A particle analysis routine was employed using a flood-fill algorithm to enhance Ca2+ transient detection. particles (PTCLs) representing the areas of active Ca2+ signals in cells were saved as a coordinate-based particle movie and combined area and total number of PTCLs were calculated. To better isolate firing sites, only those particles that did not overlap with any particles in the previous frame but overlap with particles in the subsequent 70 ms were considered firing/initiation sites. To show the overall regions in cells where Ca2+ transients occurred, PTCLs were summed throughout the video to create an image of their occurrence. The spatial information in the Ca2+ occurrence maps data is an indication of each firing site that gives information on their temporal activation and provides no information on their spatial spread. This was implemented in our analysis to accommodate a large number of firing sites and effectively plot them in a 2D occurrence map.Movies of Caeviously . Briefly3, 120.35; KCl, 1.2; MgCl2, 15.5; NaH2PO4,1.2; CaCl2, 2.5; and glucose, 11.5. The KRB solution was warmed to a physiological temperature of 37 ± 0.3°C and bubbled with a mixture of 97% O2 – 3% CO2. For experiments utilizing external solutions with 0 [Ca2+]o, CaCl2 was omitted and 0.5 mM ethylene glycol-bis (β-aminoethyl ether)-N, N, N’, N’–tetraacetic acid (EGTA) was added to the solution. NNC 55–0396 and TTA-A2 were purchased from Alomone Labs . 2-aminoethyl-diphenylborinate (2-APB), tetracaine, nicardipine, pinacidil was purchased from Millipore-Sigma . Thapsigargin, isradipine, Z-944, CPA and ryanodine were purchased from Tocris Bioscience . GSK 7975A was purchased from Aobious , and xestospongin C (XeC) was purchased from Cayman Chemical .All tissues were perfused continuously with KRB solution containing (mmol/L): NaCl, 5.9; NaHCOt-test or one-way ANOVA with a Tukey post hoc test where appropriate. In all tests, p<0.05 was considered significant. When describing data, n refers to the number of animals used in a dataset. Probabilities < 0.05 are represented by a single asterisk (*), probabilities < 0.01 are represented by two asterisks (**), probabilities < 0.001 are represented by three asterisks (***) and probabilities < 0.0001 are represented by four asterisks (****). All statistical tests were performed using GraphPad Prism 8.0.1 .Data is presented as the mean ± standard error unless otherwise stated. Statistical analysis was performed using either a Students  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Gastrointestinal motility is controlled by the activity of interstitial cells of Cajal (ICC). Here the authors elucidate the calcium signals that drive the activity of submucosal ICCs which in turn elicit phasic contractions of the colon. The authors demonstrate that the calcium signals depend on calcium entry into submucosal ICCs.Decision letter after peer review:2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the colon" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Michael J Davis (Reviewer #3).Thank you for submitting your article "CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the colon", the authors investigated the role of different Ca2+ pathways on pacemaker (slow wave) activity in the mouse colon. While much of the investigation uses techniques and tools used to previously by the authors study Ca2+ dynamics in ICC in other regions of the gut , they have refined the colonic tissue preparation to remove motion artifacts and have used dual Ca2+ indicator mice to better understand how ICC-SM influence smooth muscle Ca2+ dynamics and contraction. Overall, this solid study has used a comprehensive battery of drugs that interfere with Ca2+ signaling to demonstrate that extracellular Ca2+ entry is a particularly important component of pacemaker activity in ICC-SM.In the manuscript entitled "CaRevisions:2+ (amplitude/duration), and are likely to result in stronger/more complete activation of the SM. In terms of motility, it is possible (and likely) that "stronger" pacing at half control frequency may impart more useful force to contents and potentially improve motility. As the authors are well aware, this may be the default situation in the stomach (2pacemaker:1contraction). An analysis of a number of common parameters  performed on each observable event  would be important to better assess the effects of the drugs used.1) Paradoxically, firing failure usually causes subsequent pacemaker events to release more Ca2+ began to cause instabilities in the coordination/synchronization in site firing at 1mM while maintaining a frequency close to that observed at 2.5mM. At 0.5mM, firing failure occurred, but in the examples where firing occurred, it was the most synchronous / coordinated observed in the entire experiment. This, again, could indicate that lengthening the duration of "recovery" after a pacemaker event is one of the major factors determining the coordination of both intracellular (site) firing, and by extension, intracellular (network) activity. In contrast to the author's conclusions, this process does not appear to be that dependent on extracellular Ca2+ entry and needs to be commented on. In addition, lowering calcium will also affect membrane surface charge, which in itself will affect the electrical field experience by voltage-dependent ion channels.2) Interestingly, the authors show a different aspect of the "recovery" effect on the coordination of intracellular release site firing – which is somewhat unexpected, and important. Decreasing extracellular Ca2+ data into PTCLs based on "first" event seems arbitrary, as the degree to which Ca2+ spreads from the position of the "first" event appears to be quite variable. The image illustrating individual sites appears to be hand-drawn. If site demarcation was somewhat arbitrary, this should be clarified.3) The spatial segmentation of dynamic Ca4) The cell density value reported (95 cells per mm2) seems low by an order of magnitude. If the quoted average minimum separation distance is 30µm, one would expect 33 cells across a 1mm in 1D. Using the same separation distance, in 2D, one would expect 33 cells x 33 cells = ~1000 cells per square mm.2+ events, smooth muscle Ca2+ events and resultant micromotion  in general terms, the traces shown in Figure 2E do not show a 1:1 correlation of the event waveforms. While some variability is expected between micromotions and fluorescence signals due to unknown dynamic/static stress and strain in the gut wall, one would expect the Ca2+ event waveforms in ICC-SM and SM to be more correlated given the tight electrical coupling via gap junctions. While all the presented Ca2+ events are biphasic in nature, the first event ICC-SM shows a clear separation between the 2 peaks. The time between the 2 peaks appears sufficient to allow for re-stimulation the SM (~1s), given the fluorescence has returned 80-90% back to baseline – yet there is no inflection or change in slope in the SM Ca2+ event. The authors should comment on this disparity and qualify under which conditions (frequency/amplitude/? range) the correlation holds true using cross-correlation analysis. Differentiation and distortion vectors would also better locate contraction epicenters in videos and differentiation of traces would provide a more reliable landmark (max dI/dT) to compare time delays.5) While a correlation exists between the ICC-SM Ca6) The stomach and small intestine generate slow waves in the outermost ICC layer (myenteric layer: ICC-MY) that co-activates circular and longitudinal muscle layers. Activation of colonic CM from submucosal ICC layer prevents slow waves from directly co-activating longitudinal SM, and this is evident in more independent and varied longitudinal motions . The role of colonic ICC-MY is more confusing but appears to synchronize activity in certain conditions, which has been proposed to change the excitability of CM and possibly other layers in the gut wall. As such, without knowing the output from colonic ICC-MY, attempts to correlate CM activity to ICC-SM activity may be highly variable and unreliable. A comment on this would be helpful.2+ entry through L-type Ca2+ channels is vital to the generation of pacemaker activity is valid with regard to network activity, but the effect on intracellular release sites seems less clear. In Figure 9 A, there are 8-9 well defined "hot spots" demarcated by their white/red color (denoting how long the site was active). The cytoplasm surrounding these hotspots shows a lower occurrence of activity, consistent with an occasional propagating intracellular Ca2+ wave. Interestingly, all of the main hotspots remained after nicardipine, albeit firing at a much lower frequency. Wouldn't this suggest that release sites are not intrinsically dependent on L-type Ca2+ entry and that the L-Type channels are more involved in facilitating spread of Ca2+ through the cytoplasm?7) The conclusion that Ca2+ channel opener (BayK8644) on the ICC-SM preparation? This would yield important information regarding the role of channel inactivation/"recovery" and the importance of entrainment and limiting ectopic activity on the formation of coherent pacemaker activity.8) Have the authors used an L-type Ca2+ channel entry is equally important is L-type entry. The frequency of control activity prior to the addition of NNC  was quite high and is likely to have predisposed cells to firing failure to a much larger degree as slower control frequencies.9) Wouldn't large effect of NNC after nicardipine  suggest that T-type Ca10) Raw data ± SE was presented in the both the text and in graphical form, particularly when presenting % data. The text in the Results section or the latter parts of many figures could be reduced considerably and easier to read/ view without the repetition. This would considerably raise the impact of the work.11) The results of Pinacidil alone  could easily be removed as they didn't reach significance; at the very least, moved to the supplementary section.2+ transient firing, but this effect did not reach statistical significance” and “Reduction in the duration of Ca2+ transients was associated with a tendency for an increase in frequency, but this parameter did not reach significance”12) The effects of pinacidil on Ca channel blockers could perhaps be best illustrated by plotting the red and blue bars in Figure 11 with the normalized results in Figures 9 and 10 to illustrate any effect. “Tendency” statements that should be removed include: “Pinacidil showed a tendency toward increased Ca13) The Xestospongin modulation of the Ca store signalling in ICC-SM is not significant and probably doesn't require illustration or it should be moved to the supplementary data section.14) The qPCR analyses provide evidence for message for those two channels and the use of three different pharmacological inhibitors (for Cav3) with similar effects  support their functional expression. However, can the authors rule out the possibility that NNC 55-0396, TTA-A2 and Z-944 are producing their respective effects on calcium transients by partial inhibition of L-type channels? e.g., does a lower concentration of nicardipine produce similar effects to those agents?15) Pinacidil was used to activate KATP channels in the muscle layer, when it remained connected to the ICC-SM layer; this presumably(?) led to hyperpolarization of the ICC layer due to electrical coupling between the layers. Is that correct? (If so that assumption was not clearly stated). The differential effects of the calcium channel inhibitors before and after PIN were then used as evidence that voltage-activated calcium channels with more negative thresholds played a role when PIN was used. But shouldn't the effects of isradipine also have been tested in the presence of PIN in order to make conclusions about the contribution of Cav1.3 channels? Supporting electrophysiology data showing the extent of PIN-induced hyperpolarization in ICC-SM cells would aid in interpretation of these data.16) The authors state that ICC in general do not express KATP channels, but wasn't that analysis made for another type of ICC (ICC-MY?)? If that is so, they should check for Kir6 and SUR expression in these purified ICC-SM cells.17) Previous studies of ICC-SM in mouse have used 0.01 – 1 μm nifedipine  and Ni2+ to dissect out contributions of L-and T- type channels to the initial and plateau phases of the slow waves in these cells. That data should be discussed at least to some extent. Do the authors think that the pattern of calcium events (fast and transient vs prolonged) correspond to the initial and sustained phases of the slow waves measured in previous studies, in which it was concluded that T- and L-type calcium channels respectively contributed? Revisions:2+ (amplitude/duration), and are likely to result in stronger/more complete activation of the SM. In terms of motility, it is possible (and likely) that "stronger" pacing at half control frequency may impart more useful force to contents and potentially improve motility. As the authors are well aware, this may be the default situation in the stomach (2pacemaker:1contraction). An analysis of a number of common parameters  performed on each observable event  would be important to better assess the effects of the drugs used.1) Paradoxically, firing failure usually causes subsequent pacemaker events to release more Ca2+ firing sites in ICC-SM using particle analysis method. This analysis is based on temporal characteristics to define firing sites. The number of Ca2+ firing events is enormous and variable cycle-to-cycle, as firing sites can fire multiple times. The particle analysis method relies on binary transformation to effectively define and segment Ca2+ firing sites. Therefore, the intensity amplitude of each firing site is not available, but this binary transformation enables us to effectively quantify the large number of sites within the FOV in terms of temporal information and provides parameters of Ca2+ event size, count, duration and frequency.We analyzed the Ca2+ particle events to aid the readers to better understand the observed drug effects. The event duration data are included in updated figures  and in the Results section.Following the reviewer's suggestion, in addition to firing sites size and frequency count, we analyzed all data sets under control and drug conditions and added the duration information for Ca2+ began to cause instabilities in the coordination/synchronization in site firing at 1mM while maintaining a frequency close to that observed at 2.5mM. At 0.5mM, firing failure occurred, but in the examples where firing occurred, it was the most synchronous / coordinated observed in the entire experiment. This, again, could indicate that lengthening the duration of "recovery" after a pacemaker event is one of the major factors determining the coordination of both intracellular (site) firing, and by extension, intracellular (network) activity. In contrast to the author's conclusions, this process does not appear to be that dependent on extracellular Ca2+ entry and needs to be commented on. In addition, lowering calcium will also affect membrane surface charge, which in itself will affect the electrical field experience by voltage-dependent ion channels.2) Interestingly, the authors show a different aspect of the "recovery" effect on the coordination of intracellular release site firing – which is somewhat unexpected, and important. Decreasing extracellular Ca2+] o to 0.5 mM caused a firing failure, which is most likely due to a reduction in Ca2+ entry and effectively maintaining store refilling and CICR mechanisms. This observation confirms the role of Ca2+ entry in maintaining Ca2+ release mechanisms as indicated by the reduction of the number of Ca2+ firing sites We agree with the reviewer that reducing [Ca2+ firing sites under reduced [Ca2+] o conditions . There was no significant change in the average individual firing sites duration except when we reduce [Ca2+] o to 1 mM, we noticed a significant increase. This observed increase in Ca2+ firing duration at 50% [Ca2+] o may be providing a compensatory mechanism for pacemaker firing failure. Reducing [Ca2+] o further had no significant change in individual firing site duration but the duration of the whole Ca2+ transient cycle (CTC) was reduced, and this is in line with previous paper  that showed reducing [Ca2+] o to 0.5 mM and 0.25 mM caused a firing failure in colonic slow waves that was accompanied with a reduction in electrical slow wave duration in colonic tissues.Following the reviewer’s suggestion, we analyzed the duration of Ca2+] o was reduced. We have addressed this point in a previous paper with a series of cation replacement of Ca2+ by Mg2+ were tested on colonic electrical slow waves and found no significant differences in the responses .We agree with the reviewer that membrane surface charge could influence some experiments where the divalent ion concentration decreased as o initiates ANO1 currents and depolarization in ICC, and depolarization initiates openings of Ca2+ channels and Ca2+ entry in SMCs. We don’t anticipate that subtle changes in Ca2+ transients in one or a few ICC during a single cycle would have great influence on the Ca2+ signals in SMCs, as depolarizing currents from ICC activate Ca2+ action potentials. Thus, activation of SMCs may be independent of the exact waveforms of Ca2+ transients in ICC as long as threshold is reached. Another factor separating 1:1 waveform behavior in ICC and SMCs is the refractory properties of Ca2+ action potentials in SMC and Ca2+ in ICC are fundamentally different, causing differences in the temporal and overall “shaping” of responses. To clarify this point, we added comment on the contribution of multiple ICC to the overall SMC signals in the Discussion section.In addition, ICC demonstrate complex patterns of Ca6) The stomach and small intestine generate slow waves in the outermost ICC layer (myenteric layer: ICC-MY) that co-activates circular and longitudinal muscle layers. Activation of colonic CM from submucosal ICC layer prevents slow waves from directly co-activating longitudinal SM, and this is evident in more independent and varied longitudinal motions . The role of colonic ICC-MY is more confusing but appears to synchronize activity in certain conditions, which has been proposed to change the excitability of CM and possibly other layers in the gut wall. As such, without knowing the output from colonic ICC-MY, attempts to correlate CM activity to ICC-SM activity may be highly variable and unreliable. A comment on this would be helpful.2+ transients and activation of ANO1 channels, can contribute to colonic excitability. Both ICC-SM and ICC-MY provide pacemaker activities in the colon. To understand the complex behaviors of these cells, a reductionist approach is first needed. In this study we focused on the intrinsic pacemaker role of ICC-SM in driving adjacent smooth muscle. Most of the experiments in the study, used an isolated ICC-SM preparation to define Ca2+ signals in ICC-SM with minimal motion artifacts. To examine if ICC-SM drive activity in CM, we used a preparation that included attached SMCs. It is true these preparations included additional types of ICC that might also influence the ultimate behaviors of SMCs. Therefore, we cannot exclude the fact that the signals in SMCs under the conditions of these specific experiments are influenced by additional types of ICC. We know from this study and previously published papers that ICC-SM in the mouse colon generates slow waves at ~ 15 /min whereas ICC-MY ~2/min. Contractions observed in our experiments followed the frequency of behaviors in ICC-SM. Possible influences from additional cells within the muscle tissues were not evaluated in this study. To fully understand the highly integrated functions in colonic motility that result from the behaviors of many types of cells: ICC, SMCs and interstitial cells labeled with antibodies to PDGFRα (i.e. the SIP syncytium), enteric neural inputs and tuning of neural activity by glial cells, will require considerably more complex imaging approaches and refined techniques to limit movements without interfering with the electrical behaviors and Ca2+ dynamics of the cells involved.We agree with the reviewer that several types of ICC exist in the colon, and all, through coupling of CaAt this point, we do not know if the activity of ICC-SM propagates through the CM to influence the behaviors of ICC-MY and longitudinal muscle or vice versa. Previous studies in larger animals have shown that pacemaker propagation declines as a function of distance from the submucosal region towards the myenteric region. Smith et al., 1987 AJP.2+ entry through L-type Ca2+ channels is vital to the generation of pacemaker activity is valid with regard to network activity, but the effect on intracellular release sites seems less clear. In Figure 9 A, there are 8-9 well defined "hot spots" demarcated by their white/red color (denoting how long the site was active). The cytoplasm surrounding these hotspots shows a lower occurrence of activity, consistent with an occasional propagating intracellular Ca2+ wave. Interestingly, all of the main hotspots remained after nicardipine, albeit firing at a much lower frequency. Wouldn't this suggest that release sites are not intrinsically dependent on L-type Ca2+ entry and that the L-Type channels are more involved in facilitating spread of Ca2+ through the cytoplasm?7) The conclusion that Ca2+ release sites continued to fire throughout experiments (hot-spots). But after nicardipine these sites fired randomly, and their frequency was very low. Thus, we interpret these observations to mean that Ca2+ release is an intrinsic property of ICC, and it occurs from specific sites within cells. Ca2+ influx enhances the excitability of Ca2+ sites and is crucial for organizing Ca2+ release into clusters that cause sustained activation of ANO1 channels and electrical depolarizations (slow waves). Ca2+ entry organizes Ca2+ release via CICR. Ca2+ entry through L-type Ca2+ channels may also contribute to refilling of stores.After the addition of nicardipine a few remnant Ca2+ channel opener (BayK8644) on the ICC-SM preparation? This would yield important information regarding the role of channel inactivation/"recovery" and the importance of entrainment and limiting ectopic activity on the formation of coherent pacemaker activity.8) Have the authors used an L-type CaWe have used the L-type channel antagonist (Nicardipine) in this study to evaluate contributions of these channels, however we have not used an L-type channel agonist, such as BayK8644. It is an interesting suggestion, and we will utilize this compound in future studies.2+ channel entry is equally important is L-type entry. The frequency of control activity prior to the addition of NNC  was quite high and is likely to have predisposed cells to firing failure to a much larger degree as slower control frequencies.9) Wouldn't large effect of NNC after nicardipine  suggest that T-type Ca2+ channels are present and may contribute to pacemaker activity in ICC-SM. However, the resting membrane potential of colonic muscles is usually not at highly negative potentials . Thus, the relative importance of T-type Ca2+ channels may be reduced due to tonic inactivation of these channels. In cases of depolarized cells, L-type Ca2+ channels may dominate, as appears to happen under resting conditions in our experiments. T-type Ca2+ channels, however, can become more important if membrane potential is hyperpolarized, as indicated by our experiments when pinacidil was used to hyperpolarize cells. This shift in importance between the two conductances could be important in pathophysiological conditions associated with changes in the membrane potential. Having two conductance expressed with different voltage-dependencies provides a safety factor for maintenance of pacemaker activity, an important and novel observation of the present study.We agree with the reviewer that both L-type and T-type Ca10) Raw data ± SE was presented in the both the text and in graphical form, particularly when presenting % data. The text in the Results section or the latter parts of many figures could be reduced considerably and easier to read/ view without the repetition. This would considerably raise the impact of the work.We thank the reviewer for this suggestion, we feel that including the Raw data ± SE in the Results section would provide the reader of details on the degree of drug effects and help in tabulate the results presented.11) The results of Pinacidil alone  could easily be removed as they didn't reach significance; at the very least, moved to the supplementary section.Following the reviewer's suggestion, we removed the effects of pinacidil alone to a new supplementary figure .2+ transient firing, but this effect did not reach statistical significance” and “Reduction in the duration of Ca2+ transients was associated with a tendency for an increase in frequency, but this parameter did not reach significance”12) The effects of pinacidil on Ca channel blockers could perhaps be best illustrated by plotting the red and blue bars in Figure 11 with the normalized results in Figures 9 and 10 to illustrate any effect. “Tendency” statements that should be removed include: “Pinacidil showed a tendency toward increased CaFollowing the reviewer's suggestion, we removed the effects of pinacidil alone to a new supplementary figure. We also removed all tendency statements from the Results section.13) The Xestospongin modulation of the Ca store signalling in ICC-SM is not significant and probably doesn't require illustration or it should be moved to the supplementary data section.Following the reviewer's suggestion, we removed the effects of Xestospongin into a new supplementary figure .14) The qPCR analyses provide evidence for message for those two channels and the use of three different pharmacological inhibitors (for Cav3) with similar effects  support their functional expression. However, can the authors rule out the possibility that NNC 55-0396, TTA-A2 and Z-944 are producing their respective effects on calcium transients by partial inhibition of L-type channels? e.g., does a lower concentration of nicardipine produce similar effects to those agents?2+ channel.A previous report has examined the selectivity of these drugs. Ming li et al. 2005 (DOI: 10.1111/j.1527-3466.2005.tb00164.x) demonstrated that 100 microM NNC 55-0396 has no detectable effect on high voltage-activated currents and does not produce metabolite that causes inhibition of L-type Ca15) Pinacidil was used to activate KATP channels in the muscle layer, when it remained connected to the ICC-SM layer; this presumably(?) led to hyperpolarization of the ICC layer due to electrical coupling between the layers. Is that correct? (If so that assumption was not clearly stated). The differential effects of the calcium channel inhibitors before and after PIN were then used as evidence that voltage-activated calcium channels with more negative thresholds played a role when PIN was used. But shouldn't the effects of isradipine also have been tested in the presence of PIN in order to make conclusions about the contribution of Cav1.3 channels? Supporting electrophysiology data showing the extent of PIN-induced hyperpolarization in ICC-SM cells would aid in interpretation of these data.In the current study, pinacidil application was used to produce a membrane hyperpolarization in SMCs via activation of KATP channels and ICC as they are electrically coupled. We added a statement in the Results section to clarify the rationale of pinacidil application.2+ transients depend upon Ca2+ influx via L-type Ca2+ channels, and since isradipine had nearly the same or a lesser effect on Ca2+ transients than nicardipine, CaV 1.2 appear dominant as the Ca2+ entry mechanism. This set of experiments were targeted to determine the contributions of T-type Ca2+ channels. The results demonstrate that at negative potentials, where the open probability of L-type Ca2+ channels is low, T-type Ca2+ channels are available to facilitate Ca2+ influx and sustain pacemaker activity. We agree that patch clamp experiments would further support the conclusions, and we will incorporate them in future studies.We agree with the reviewer point that these experiments do not directly incorporate potential involvement of Cav 1.3 channels. Data in this study show that ICC-SM Ca16) The authors state that ICC in general do not express KATP channels, but wasn't that analysis made for another type of ICC (ICC-MY?)? If that is so, they should check for Kir6 and SUR expression in these purified ICC-SM cells.ICC express message for Kir6 and SUR, however KATP currents cannot be resolved in colonic ICC . At present it is unknown whether Kir6 and SUR are not translated or not trafficked to the plasma membranes to create functional KATP channels in ICC.17) Previous studies of ICC-SM in mouse have used 0.01 – 1 μm nifedipine  and Ni2+ to dissect out contributions of L-and T- type channels to the initial and plateau phases of the slow waves in these cells. That data should be discussed at least to some extent. Do the authors think that the pattern of calcium events (fast and transient vs prolonged) correspond to the initial and sustained phases of the slow waves measured in previous studies, in which it was concluded that T- and L-type calcium channels respectively contributed?2+ channels contribute to slow waves in the colon. L-type Ca2+ channels completely abolished slow waves where T-type channels are involved in the initial component of the slow wave. Our study highlighting the Ca2+ signaling in ICC-SM is consistent with conclusions of the Yoneda et al. papers. Although the comparison between the electrical activity and Ca2+ dynamics from different studies is difficult to clearly interpret. Remember that the electrical activity was recorded (most likely) from SMCs and we are imaging Ca2+ dynamics in most experiments exclusively from ICC. Figure 15 summarizes the sequence of events in ICC, and these events drive the depolarization-dependent activation of Ca2+ action potentials/ Ca2+ entry in SMCs. We have taken the reviewer’s suggestion and added more information about the conclusions of Yoneda et al. in the Discussion.Both of Yoneda et al. papers  concluded that; L-type and T-type Ca


The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.The essential role of ORAI1 channels in receptor-evoked Ca 2+ signaling is well understood, but the roles of ORAI2 and ORAI3 remained obscure. Here authors show that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation.The essential role of ORAI1 channels in receptor-evoked Ca In non-excitable cells, stimulation of various PM receptors leads to the production of the diffusible second messenger inositol 1,4,5-trisphosphate (IP3)4. IP3 binds to the IP3 receptor (IP3R) channels5 in the endoplasmic reticulum (ER) and induces the release of ER luminal Ca2+ into the cytosol, resulting in ER store depletion. It is clearly established that ER store depletion causes a conformational change in the Ca2+ sensing protein, stromal interacting molecule (STIM), which translocate to ER–PM junctions to trap and activate ORAI1 channels and induce store-operated Ca2+ entry (SOCE)10. When ectopically co-expressed with STIM1, the ORAI1 homologs ORAI2 and ORAI3 can clearly mediate SOCE and its biophysical manifestation, the Ca2+ release-activated Ca2+ current (ICRAC)12. However, the contribution of ORAI2 and ORAI3 to native SOCE and ICRAC remains uncertain.Cytosolic calcium (Ca2+ ATPase (SERCA) blocker or high agonist concentrations  in HEK293 cells), both of which maximally deplete ER Ca2+ stores13. Under these conditions of maximal store depletion, the Ca2+ signal typically generated is dominated by a sustained cytosolic Ca2+ plateau phase14. In reality, such monotonic Ca2+ signals represent only a narrow window of the repertoire of Ca2+ signals stimulated by physiological receptor stimulation. In most in vivo settings, cells respond to much lower concentrations of tissue and circulating agonists15. In fact, at these low range  or mid-range  concentrations, Ca2+ signals in most cells are typically shaped as transient regenerative oscillations or spikes. The frequency of these Ca2+ oscillations increase with increasing agonist concentrations until they “tetanize” into plateaus at high agonist concentrations20.To reliably measure SOCE function with fluorescent dyes, most studies have relied on protocols that cause maximal SOCE activation. This is achieved by the use of either pharmacological tools such as thapsigargin, which is a sarcoplasmic/ER Ca2+ oscillations in most cells, but not all, are the direct result of Ca2+ release through IP3R, they last only for one to three spikes before running down when extracellular Ca2+ is omitted or when SOCE is inhibited19, highlighting the crucial role of Ca2+ entry in sustaining long-term Ca2+ oscillations and gene transcription. It is thought that specific frequency signatures of Ca2+ oscillations are decoded by downstream effector proteins to selectively activate specific isoforms of transcription factors, including those of nuclear factor of activated T-cells (NFAT). Furthermore, despite the fact that Ca2+ oscillations originate from ER release, there is compelling evidence that the activation of certain gene programs, specifically NFAT-dependent genes, is controlled by Ca2+ trickling from ORAI channels21. Thus, spatially restricted Ca2+ signaling microdomains at the mouth of ORAI channels exert a crucial control over cellular physiology.Although Ca2+ oscillations. We show that native ORAI3 and ORAI2 are sufficient to sustain regenerative Ca2+ oscillations but do not significantly contribute to Ca2+ plateaus, which are predominantly supported by ORAI1. This ORAI isoform-dependent behavior is determined by the magnitude of each isoform interaction with STIM1 under unstimulated conditions matched by the strength of fast Ca2+-dependent inactivation (CDI) of each isoform, with ORAI3 being the highest, ORAI2 the intermediate, and ORAI1 the lowest. In the absence of ORAI2 and ORAI3, the physiological Ca2+ responses to low- and mid-range agonist concentrations manifest mostly as plateaus, effectively shrinking the bandwidth of agonist-evoked Ca2+ signals. We also show that both ORAI2 and ORAI3 negatively regulate ORAI1 by enhancing the channel CDI through heteromerization. Our data suggest that the ORAI isoform trio functions as a heteromultimer to form the native CRAC channel. Furthermore, we show that Ca2+ entry through ORAI1 alone is necessary and sufficient to drive nuclear translocation of both NFAT1 (which requires robust Ca2+ to activate) and NFAT4 (which requires modest Ca2+ to activate), whereas ORAI2 and ORAI3 either alone or together do not support NFAT1 and NFAT4 nuclear translocation. However, ORAI2 and ORAI3 negatively regulate ORAI1-dependent NFAT1/4 nuclear translocation, in further support of native ORAI isoform heteromerization. The major consequence of ORAI2- and ORAI3-negative regulation of CRAC channels is the differential nuclear translocation of NFAT1 and NFAT4 in response to distinct agonist strengths and cytosolic Ca2+ concentrations. Our findings identify the signaling functions of ORAI2 and ORAI3. Native ORAI2 and ORAI3 heteromerize with ORAI1 to form the native CRAC channel and are required for the graded diversity of Ca2+ signaling events covering the full range of agonist concentrations/strengths. This broadened bandwidth of Ca2+ signals ensures the precise nuclear translocation of NFAT isoforms of transcription factors in response to their specific range of agonist strengths and cytosolic Ca2+ concentrations.To determine the signaling functions of native ORAI3, ORAI2, and ORAI1, we generated multiple single, double, and triple ORAI isoform knockouts in HEK293 cells using CRISPR/Cas9 technology. Using this system, we show that although ORAI1 is required for SOCE activated by maximal store depletion, surprisingly it is dispensable for Ca21 and Supplementary Table 2+ oscillations, including both STIM isoforms  to remove a large portion of the ORAI gene. Hence, the absence of the specific mRNA is a reliable means for documenting knockout. To rule out off-target effects, we generated several independent clones for each specific ORAI isoform. For each condition shown hereafter, we show data from these two independent clones. ORAI1 protein-knockout was documented with genomic sequencing and by western blotting  AgMid, 10–30 µM Cch; and (3) AgHigh, 30–100 µM Cch. We first stimulated wild-type (WT) HEK293 cells with 10 µM Cch to elicit Ca2+ oscillations in the presence of 2 mM extracellular Ca2+. Traces from five representative cells are shown in Fig. 2+ oscillations that lasted for the duration of the recording, with an average of 6 oscillations/14 min  Agmin Fig. . Of the min Fig. .Fig. 1SO3+) in the bath , which block both Ca2+ entry and Ca2+ extrusion  in ORAI-TKO cells restores their oscillatory behavior to levels of WT HEK293 cells  showed almost complete abrogation of SOCE in response to thapsigargin  as universally reported in essentially all cell types  cells failed to sustain oscillations and displayed a similar behavior to that of WT HEK293 cells in which SOCE was blocked by inclusion of a low concentration of lanthanides  cells, namely ORAI2,3-DKO, ORAI1,3-DKO, and ORAI1,2-DKO cells. SOCE in response to thapsigargin is abrogated in ORAI1,2-DKO and ORAI1,3-DKO cells Fig. , highliglls Fig.  and thislls Fig. , suggestlls Fig. . Cells wing Fig. .2+ oscillations . To gain better insights into ORAI isoform contribution in response to a wide range of agonist concentrations, we measured Ca µM Fig. , focusin µM Fig. , with OR µM Fig. . As for  µM Fig. . For com2+ oscillations from five different cells/condition for the double ORAI-knockout cells  and the ORAI-TKO cells are shown in Fig. 2+ spike in response to each increasing concentration of agonist  knockdown of ORAI1 in HEK293 cells and proposed that ORAI1 is required for mediating Caons Fig. . We thenity Fig. . Our datity Fig. , matchinity Fig. . HoweverRNA Fig. . ORAI1 kRNA Fig. , an incrRNA Fig. , and redRNA Fig. . These r12. To obtain physiologically meaningful results that relate to native conditions of expression, we expressed each individual ORAI isoform in ORAI-TKO cells using plasmids that achieve expression levels similar to the native condition. These plasmids were generated by sub-cloning the yellow fluorescent protein (YFP)-tagged cDNA of each ORAI isoform under the control of the “weak” thymidine kinase (tk) promoter26. Hence, we circumvented the need to overexpress STIM1 and relied on native STIM proteins to activate these ORAI isoforms . Based on YFP fluorescence of tagged ORAI isoforms, we analyzed only cells with comparable levels of YFP fluorescence  promoter), the magnitude of SOCE activated by thapsigargin is as follows: ORAI1 > ORAI2 > ORAI3nce Fig. . We woulnce Fig. .Fig. 3ORIn response to 10 µM Cch, each ORAI isoform was capable of supporting oscillations in individual cells with frequencies of oscillations marginally higher in ORAI2- and ORAI3-expressing ORAI-TKO cells than ORAI1-expressing cells Fig. . Signifi2+ oscillations, we tested whether STIM1 preferentially interacts with ORAI2 and ORAI3 over ORAI1. We reasoned that a potential privileged interaction of ORAI2 and ORAI3 with STIM1 would allow these two ORAI isoforms to be activated by lower levels of store depletion, such as those evoked by low agonist concentrations. Interestingly, Förster resonance energy transfer (FRET) measurements of STIM1-YFP co-expressed with either CFP-ORAI isoform in ORAI-TKO cells showed that ORAI2 and ORAI3 have a significantly higher basal FRET with STIM1 compared with ORAI1 12. These differences in fast CDI between ORAI isoforms were confirmed in our co-expression studies, now performed in ORAI-TKO cells and showing that CDI of ORAI3 > ORAI2 > ORAI1, whereas peak whole-cell currents follow an inverse relationship of ORAI1 > ORAI2 > ORAI3  are shown in the middle panels while data summary of current densities from several recordings are shown in the lower panels. We exploited the different pharmacological sensitivity of ORAI isoforms to the compound 2-aminoethoxydiphenyl borate (2-APB), whereby at 50 µM 2-APB, ORAI1 is completely inhibited, ORAI2 is only partially inhibited, and ORAI3 is potentiated31 . However, in ORAI-SKO and ORAI-DKO cells, CRAC currents are only observed in cells expressing ORAI1  bath solutions to amplify these currents in WT HEK293 cells and ORAI-KO clones  is equivalent to agonist concentration over time (s). Figure 2+. The response to thapsigargin is simulated by decreasing the SERCA pump flux to zero. The differences between ORAI2 and ORAI3 in their negative regulation of ORAI1 and Ca2+ oscillations cannot be distinguished by the model, and as such the model predictions depicted in Fig. 2+ responses predicted in response to increasing concentrations of agonist generate plateaus earlier in ORA2,3-DKO cells  stimulation as previously described -tagged NFAT reporter constructs (NFAT1-GFP and NFAT4-GFP) in our parental WT HEK293 cell line and in each of our single, double and triple ORAI clones and then stimulated these cells with 10 µM Cch. Nuclear translocation of each NFAT-GFP fusion protein was recorded over time by monitoring the ratio of nuclear to whole-cell GFP fluorescence . Further, 2-APB-mediated activation of ORAI3 is only apparent in cells lacking ORAI2 and/or ORAI1. These results strongly argue that native ORAI isoforms heteromerize with one another and that through this process, ORAI2 and ORAI3 associations with ORAI1 regulate CRAC channel activity to produce Ca2+ signals that match the diversity of agonist strengths. Indeed, STIM1 co-expression with two different ORAI isoforms or with ORAI concatenated heterodimers generates CRAC channels with an intermediary peak current and CDI. Furthermore, our mathematical modeling lend strong support to ORAI isoform heteromerization. Our model could reproduce the Ca2+ oscillations data obtained experimentally only when: (1) ORAI2 and ORAI3 associate preferentially with ORAI1, rather than with themselves; (2) the ORAI1,2 and ORAI1,3 heteromers allow substantially less Ca2+ current than does the ORAI1,1 homomer; and (3) that cells express approximately two- to fivefold more of ORAI1 proteins than ORAI2 or ORAI3 proteins. These predictions are consistent with the experimental data.Our ORAI rescue experiments in ORAI-TKO cells suggest that ORAI2 and ORAI3 levels in HEK293 cells are significantly less than those of ORAI1. From our various ORAI isoform knockout combinations, it is clear that each ORAI isoform can function alone under native conditions to support Ca2+ signaling profile. If the position of an ORAI isoform within the hexameric channel is not biologically relevant and only the number of each ORAI isoform matters , then there are 28 potential hexameric channel combinations that can be easily calculated: 3 homohexamers; 5 heterohexamers for each 2 isoform combinations  and 10 heterohexamers for 3 isoform combinations. If the position of each isoform within the hexamer is important, then the number of total hexameric combinations is 92 based on Burnside’s Lemma calculations with 3 homohexamers, 33 heterohexamers for 2 isoform combinations (11 for each 2 ORAI isoform combinations), and 56 heterohexamers for 3 isoform combinations. . Transfections were performed using the Amaxa Nucleofector II  per the manufacturer instructions. In brief 1 × 106 cells were resuspended in solution V and the cDNA plasmid added to the cell suspension. Once mixed cells were placed in a standard cuvette, transfected, and resuspended in complete DMEM media. Once resuspended, cells were transferred to 25 mm glass coverslips and returned to the incubator for 24 h prior to experimentation.Parental HEK293 cells were obtained from ATCC  and maintained in high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1× Antibiotic–Antimycotic solution and 10% heat-inactivated fetal bovine serum. Cells were maintained in a humidified COTwo methods were used to generate ORAI-KO HEK293 cell lines. The first method utilized a single ORAI specific gRNA subcloned into the lentiCRISPR v2 vector  to generate insertion/deletions within the ORAI gene. Wild-type HEK293 were transfected with the corresponding ORAI1 or ORAI3 targeting vector using nucleofection as described above. Two days after transfection, puromycin (2 μg/ml)  was added to the medium to select for cells expressing the lentiCRISPR v2 vector. Six days after puromycin selection, cells were collected and seeded at a density of 1 cell/well into 96-well plates. Once colonies had formed clones were screened using the Guide-it Mutation Detection Kit  and genetic knockout confirmed by Sanger sequencing. This method was used to generate both ORAI1-SKO clones (#1 and #6) and ORAI3-SKO clone #25. All gRNA sequences used to generate ORAI-KO cell lines can be found in Supplementary Table The second method was later adapted to achieve genomic deletion of ORAI2 and ORAI3 in the remaining set of SKO, DKO, and TKO clones. We subcloned two ORAI2 or two ORAI3 specific gRNAs flanking the entire gene into two fluorescent vectors (pSpCas9(BB)-2A-GFP and pU6-(BbsI)_CBh-Cas9-T2A-mCherry: Addgene). Twenty-four hours after transfection, single cells with high expression of GFP and mCherry were sorted into 96-well plates (FACS Aria SORP high-performance cell sorter). Cells were then maintained in complete medium until colonies began to form. Visible colonies were collected, DNA extracted, and screened using specific primers designed to resolve a WT or knockout PCR product. Positive clones were chosen when only the knockout band was seen with no product in the WT reaction. The complete list of primers and gRNAs can be found can be found in Supplementary Tables Novel constructs including the ORAI2,3 concatenated heterodimer, all tk-driven ORAI1/2/3 constructs, CRISPR gRNAs, and ORAI dominant negative pore mutants were generated using primers compatible with the in-fusion cloning system  under standard cell culture conditions. After 30 min, excess Fura2 AM was washed away and coverslips were mounted in an Attofluor cell chamber. Prior to recording, cells were washed three times with 2 mM Ca2+ Hepes-buffered salt solution  supplemented with 2 mM Ca2+, as described previously49. Fura2 AM was alternately excited at 340 nm and 380 nm using a fast shutter wheel (Sutter Instruments) and the resulting 510 nm emission was collected from individual cells on a pixel by pixel basis through a ×20 fluorescence objective using a Hamamatsu Flash 4 camera and processed using Leica Application Suite X software.Twenty-four hours before cell imaging, 1.5 × 10Whole-cell and nuclear NFAT1/4-GFP (Addgene) fluorescence was measured using an ×40 oil-immersion objective paired with a Hamamatsu Flash 4 camera and processed using the Leica Application Suite X software. NFAT1/4-GFP was excited using a 488 nm fast wheel filter and emission spectra selectively captured through a GFP filter cube. Using the equation50. All other fluorophores were imaged using the same system and corresponding excitation filters.NFAT nuclear translocation was quantified as a function of time in response to 10 µM Cch as previously describedIDD, IAA, and IDA were the respective fluorescent intensities of CFP, YFP, and FRET channels, respectively, after background subtraction. Calculation of E-FRET was performed as previously described using the formula49Images were collected at room temperature using a Leica DMI 6000B inverted automated fluorescence microscope equipped with a ×40 oil-emersion objective lens . At each time point three images were sequentially collected allowing for simultaneous quantification of CFP, YFP, and FRET fluorescence, respectively, using SlideBook 6.0 software (Intelligent Imaging Innovations). Exposure times were fixed across experiments at 250 ms (YFP), 1000 ms (CFP), and 1000 ms (FRET). Three channel FRET was calculated using the formulaTo determine FRET between two ORAI isoforms, CFP/YFP-tagged ORAI constructs were transfected with STIM1-mCherry into ORAI-TKO HEK293. To achieve comparable levels of fluorescence for all ORAIs, 3, 4, and 5 µg of ORAI1, ORAI2, and ORAI3, respectively, were used. CFP, YFP, and FRET images were collected from cells with similar CFP/YFP ratios at baseline and 5 min after treatment with either increasing doses of Cch  or after maximal store depletion with 1 µM ionomycin.Similar constructs and methods were used to determine FRET between CFP-tagged ORAI isoforms and STIM1-YFP. To achieve comparable YFP/CFP ratios, CFP-ORAI1 (2 µg), CFP-ORAI2 (2 µg), and CFP-ORAI3 (6.5 µg) were co-expressed with (1.5 µg) of STIM1-YFP. In these experiments, FRET was measured every 20 s for 10 min.g for 12 min at 4 °C and clarified supernatant collected for measurement of protein concentration. Using the Pierce BCA assay, protein extracts were quantified prior being loaded into a NuPAGE 4–12% Bis-Tris precast protein gels. Migrated proteins was transferred to polyvinylidene difluoride membranes using the transblot turbo system (Biorad). Membranes were then blocked with LI-COR Tris-buffered saline (TBS) buffer for 1 hr at room temperature and incubated with primary antibody overnight at 4 °C. Probed membranes were washed with TBS with Tween (TBST) then probed while shaking with secondary antibodies for 1 h at room temperature. Resulting blots were imaged and quantified using a LI-COR odyssey imaging system running Image Studio software (LI-COR).Cells were counted using trypan blue exclusion then washed with ice-cold phosphate-buffered saline before lysis in ice-cold RIPA buffer  supplemented with protease and phosphatase inhibitor  for 30 min. Lysates were centrifuged at 15,000 × 6) were lysed in TRIzol (Invitrogen) and total RNA extracted according to the manufacturer protocol. Isolated RNA was quantified using a nanodrop 2000 spectrophotometer (Thermo Scientific) and 1 µg of RNA used to generate cDNA using the High-Capacity cDNA Reverse Transcription Kit according to the manufacturer’s protocol (Applied Biosystems). Total cDNA (1 µL) was then added to SYBR Green qPCR Master Mix (Applied Biosystems) and the corresponding primers added resulting in a 10 µL reaction. Samples were then loaded into a 96-well plate and data from the resulting reaction was collected and quantified using the QuantStudio 3 real-time PCR system (Applied Biosystems). The PCR protocol for all reactions began with an initial activation 2 min 50 °C step followed by a 95 °C 2 min melt step. The initial steps were followed by 40 cycles that began at 95 °C for 15 s followed by 15 s at 54.3 °C and 72 °C for 30 s. After completion of 40 cycles, a standard melt curve was generated to ensure primer specificity. All primer sequences used are provided within Supplementary Table HEK293 Cells  the first group (oscillating cells) represents cells that display regenerative oscillations for the duration of the experiment, where each oscillation returns to baseline before the start of the next oscillation. (2) The second group (non-responders) are cells that show either no response at all to agonist stimulation or showed only one initial spike and remained at baseline for the duration of the recording. (3) The third group (plateau cells) sustained a cytosolic Ca2+ signal that was ≥25% of the initial peak, for at least 5 min after stimulation  and 1 µg of one either CFP-tagged ORAI isoform or 1 µg of tdTomato-tagged ORAI heterodimer. For experiments with co-expression of two ORAI isoforms, 0.5 µg of each CFP-ORAI isoform was co-transfected with 4 µg of YFP-STIM1.2 was included in the pipette solution to inhibit TRPM7 currents. The Clampex 10.3 software was used for data collection and Clampfit 10.3 software (Molecular Devices) was used for data analysis.Cells were seeded onto 30 mm glass coverslips 24 h before recordings. Traditional patch-clamp electrophysiological recordings were performed using an Axopatch 200B and Digidata 1440A (Molecular Devices). Pipettes were pulled from borosilicate glass capillaries (World Precision Instruments) with a P-1000 Flaming/Brown micropipette puller (Sutter Instrument Company) and polished using a DMF1000 (World Precision Instruments). The resistances of all filled pipettes ranged from 2 to 4 MΩ. Recordings were performed only on cells forming tight seals (>16 GΩ) and only when the whole-cell configuration generated less than 8 MΩ values of series resistances. As a precaution, 8 mM MgCl4, 10 mM Hepes, 20 mM CaCl2, and 10 mM glucose (pH 7.4 with NaOH). The pipette solution contains 135 mM Cs-methanesulfonate, 10 mM EGTA, 8 mM MgCl2, and 10 mM Hepes (pH 7.2 with CsOH).All CDI recordings were measured from ORAI-TKO cells co-expressing STIM1 and ORAI constructs driven by CMV promotors. STIM1 and ORAI constructs used for CDI were transfected at a 4 µg (STIM1) to 1 µg (ORAI) ratio. For ORAI isoform co-transfections, 0.5 µg of each ORAI cDNA was transfected totaling 1 µg ORAI cDNA per condition. Expression of both STIM1 and ORAI constructs was verified by fluorescence microscopy. Immediately after break-in and before CRAC currents had developed, families of 150 ms voltage steps  were run with 2 s intervals between steps. At the beginning of each pulse, a 2.5 ms voltage step to 0 mV was used to eliminate residual capacitive artifacts. The first voltage step from each recording was subtracted from the resulting trace as leak current. Next, a 250 ms voltage ramp from +100 to −140 mV was administered every 2 s until CRAC currents reached steady-state level . Once steady state was achieved, CDI was determined by performing a second round of voltage steps, using the same protocol as described above. CDI was quantified as the remaining current measured at 146 ms from the peak current at the beginning of the pulse. The bath solution contains 115 mM Na-methanesulfonate, 10 mM CsCl, 1.2 mM MgSOI/V curves obtained in DVF bath solutions, representing the maximal background current, are averaged and then subtracted from the average I/V of BAPTA- or 2-APB-activated Na+ currents obtained in DVF bath solutions when the current is maximal. These subtracted I/V curves are represented as independent I/V curves in all figures. The bath solution contains 115 mM Na-methanesulfonate, 10 mM CsCl, 1.2 mM MgSO4, 10 mM Hepes, 20 mM CaCl2, and 10 mM glucose (pH 7.4 with NaOH). The pipette solution contains 115 mM Cs-methanesulfonate, 20 mM Cs-BAPTA, 8 mM MgCl2, and 10 mM Hepes (pH 7.2 with CsOH). The DVF solution contains 155 mM Na-methanesulfonate, 10 mM HEDTA, 1 mM EDTA, and 10 mM Hepes (pH 7.4 with NaOH).Recordings of native CRAC currents were performed on WT HEK293, ORAI-SKO, ORAI-DKO, and ORAI-TKO cell lines. Cells were maintained at a +30 mV holding potential during experiments and subjected to reverse voltage ramps from +100 to −140 mV lasting 250 ms at 2 s intervals. Immediately after establishing the whole-cell configuration, a 30 s DVF pulse ethane-N,N,N′,N′-tetraacetic acid (BAPTA)) was applied to gauge maximal background current. The 2+ dynamics model is an adapted version of that in ref. 22. In brief, the cell is assumed to be well-mixed and the IP3Rs are modeled by a deterministic model51 that was originally based on the stochastic model of ref. 52. STIM are not modeled explicitly and thus ORAI activation is an algebraic function of ER [Ca2+].The Ca22 is the modeling of the Ca2+ influx across the PM, which we do by a simple ORAI binding model. ORAI1 (O1), ORAI2 (O2), and ORAI3 (O3) bind to form dimers according to the reactionsThe major difference from ref. I1, I2, and I3 of ORAI1, ORAI2, and ORAI3, respectively. We set I1 = 1, I2 = 0.2, and I3 = 0.2. Only the relative values matter. I2 and I3 can be increased to around 0.5 without making any significant qualitative difference.Assume that the unstimulated cell starts with initial amounts, We have the equilibrium equationsFurthermore, conservation of ORAI givesOij has a conductance of αij and so the total Ca2+ influx through the population of dimers, Jin, will be given byFinally, we assume that each Jin. Thus, to get a model oscillation with the same period as that observed experimentally  and and18) aSome of the equilibrium constants can be determined algebraically; this reduces complexity and imposes explicit constraints on some parameter values.K11, we have to solve the two equationsTo find the equilibrium constants for the DKO cases: to calculate K11 between these two equations to getWe eliminate K22 and K33, with analogous constraints on d11, d22, α11, and α22.The same approach gives us O1, O2, and K12. There is now no simple algebraic solution. Instead, we take a numerical approach. We vary K12 from, say, 1–500, and for each value we calculate O1 and O2 from Eqs. 32\documeK12. This entire procedure can be incorporated in a continuation algorithm so that K12 can be found as a function of the parameters αij; thus, the regions of parameter space for which a solution exists can be determined. For example, for the model we used here, solutions for K12 and K13 exist only when α11/α12 > ~20/3 and α11/α13 > ~20/3.If no root exists, then it is not possible to solve for This implies that the model is consistent with the data only when ORAI2 and ORAI3 inhibit ORAI1.dij, which are highly model-dependent. As yet, it is not clear how to incorporate model variability into the parameter estimation method.So far we have used Eqs. –18) and and18) aK11 = 14, K22 = 1.5, K33 = 0.93, K12 = 19, K13 = 17, K23 = 14, all in units of 1/concentration. Again, the actual values are unimportant; only the ratios have meaning. These values imply that ORAI2 and ORAI3 preferentially bind to ORAI1.The result is I1 = 1, I2 = 0.2, I3 = 0.2 , K11 = 14, K22 = 1.5, K33 = 0.93, K12 = 19, K13 = 17, K23 = 14 , and α11 = 2, α22 = 1, α33 = 0.7, α12 = 0.1, α13 = 0.1, α23 = 0.1 . It is noteworthy that the absolute values are not important, only the ratios of the values.Typical simulations of the model are shown in Fig. p) for the WT cells and for a selection of ORAI knockouts. In every case, an increase in agonist causes an increase in oscillation frequency, a slightly raised baseline to the oscillations, and, eventually, termination of the oscillations in a raised plateau. Panel a shows that when ORAI1 and ORAI2 are knocked out, the oscillation frequency decreases dramatically, but that ORAI3 is still capable of mediating sustained oscillations. Panel b shows that the ORAI1-SKO case has greatly decreased oscillation frequency, while the ORAI2-SKO case has a frequency only slightly decreased from the WT case in panel a. Panel c shows that when all the ORAI are knocked out, oscillations can still be sustained for a small number of peaks, but that when only ORAI2 and ORAI3 are knocked out, oscillation frequency increases from WT, and the plateau happens at lower agonist concentrations.Figure 2+, and then, once the cell was emptied of Ca2+, external Ca2+ was reapplied. The resultant Ca2+ influx is a measure of the CRAC current. The ORAI1,2-DKO current (which is current only through ORAI3) is extremely small, but is still large enough to support sustained oscillations, as shown in panel a. Conversely, the ORAI2,3-DKO current is much larger than the WT current, which implies that there is less Ca2+ influx when ORAI2 and ORAI3 are present also.Panel d shows the model simulation of experiments in which thapsigargin was applied to the cell in the absence of external Ca2+ influx through ORAI1, (2) very small Ca2+ entry through ORAI3 channels is sufficient to support Ca2+ oscillations.Taken together, these model results suggest that (1) ORAI2 and ORAI3 inhibit CaAccording to Burnside’s Lemma, the number of distinct three colorings of the six cycle ismentclasspt{minimaIt follows that the number of distinct three colorings isReplacing base 3 in the above expression with either 1 or 2 yields the number of distinct one or two colorings for a given single color or pair of colors, respectively, i.e.,Thus, by the inclusion/exclusion principle the total number of three colorings that use all three colors is: 92 − (3 × 13) + (3 × 1) = 56.t-test was used. If greater than two groups were compared, then One-way analysis of variance was used. For all results with normally distributed data, parametric statistical tests were used. When data were not normally distributed, non-parametric statistical tests were used. Data normality was determined using Prism 8.0. All data are represented as the mean and error reported as ±SEM. Throughout the manuscript *, **, ***, and **** indicate p-values of <0.05, <0.01, <0.001, and <0.0001, respectively. All data with p < 0.05 was deemed significant; “ns” = nonsignificant. The exact p-values for each statistical comparison performed in the study are provided as an excel file (Supplementary Data Statistical tests were performed using GraphPad Prism 8 and Origin 9.0 software. When comparing two groups the Student’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 2Reporting Summary