TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the ~3 Å resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post-filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2.Transient receptor potential melastatin 2 (TRPM2) is a Ca TRPM2 belongs to the M (Melastatin) subfamily of transient receptor potential (TRP) ion channels. Despite a high degree of sequence conservation within the subfamily, its eight members are involved in a multitude of biological processes, and are regulated by diverse stimuli . TRPM2 p2+ permeable non-selective cation channel opened by simultaneous binding of three obligate co-activators, cytosolic ADP-ribose (ADPR) (2+ (2) (2+ are debated (2+ and PIP2 are interrelated (TRPM2 is a Cae (ADPR) , Ca2+ (CDPR)  . The loc debated , and regrrelated  through 2+. However, as TRPM4 is monovalent selective, its activation requires a nearby Ca2+ source, such as a co-localized Ca2+ channel , a TRP domain, and a coiled-coil. In TRPM2 a C-terminal ~270 residue NUDT9-homology (NUDT9H) domain serves to bind ADPR . So far, channel . In contfeedback . Furtherfeedback , but do feedback .Nematostella vectensis (nvTRPM2) in complex with Ca2+ by electron cryo-microscopy (cryo-EM), at a resolution of 3.1 Å, and correlated the structure with detailed biophysical characterization of channel permeation and gating properties. Although the global architecture of nvTRPM2 is similar to that of TRPM4, distinct local structural features provide explanations for permeation and gating properties that are unique to TRPM2, in particular to its regulation by intra- and extracellular Ca2+ as well as by PIP2.Here, we determined the structure of the TRPM2 channel from the cnidarian Xenopus laevis oocytes, and studied their biophysical properties in inside-out cell-free patches, under rapid continuous superfusion of the cytosolic membrane surface (colored bars). In the absence of extracellular (pipette) Ca2+ ([Ca2+]o buffered to ~1 nM), large macroscopic nvTRPM2 currents could be activated by superfusion of the cytosolic patch surface with ADPR  and Ca2+ . ADPR and intracellular Ca2+ acted as obligate co-activators, as addition of both ligands was required to open, whereas removal of either ligand was sufficient to close the channels (2+was only modestly (by 10–20%) enhanced upon exposure to exogenous PIP2 (left blue bar), it was almost completely abolished when membrane PIP2 headgroups were masked with polylysine (red bar), but could then be fully recovered by addition of exogenous PIP2 (right blue bar). Thus, PIP2 is also essential for nvTRPM2 activity, but is tightly bound, so that the channels remain close-to-saturated even at the low PIP2 concentration of an inside-out patch. All in all, gating regulation by ADPR, Ca2+, and PIP2 of nvTRPM2 is very similar to that of hTRPM2 (The nvTRPM2 protein shows ~63% sequence similarity (34% sequence identity) with human TRPM2 (hTRPM2), and when expressed in mammalian cells, it forms functional channels opened by cytosolic ADPR . Becausechannels . Althouglylysine , red barf hTRPM2 .2+ and absence of ADPR. , a small ankyrin repeat domain consisting of two helical hairpins , a linker helix domain , and a Pre-S1 helix (orange). Like in all known TRP channel structures, the first four transmembrane helices assemble into a voltage sensor-like domain  and the last two form the pore domain . TRP helix 1 is an extension of helix S6, and together with TRP helix 2 and an unstructured loop (TRP loop) forms the TRP domain (red). At the C terminus a ‘stretcher’ helix (pink) is followed by a coiled-coil helix  (The nvTRPM2 structure is shaped like a square prism with dimensions of 135 x 100 x 100 Å ( purple) .Bottom tier) which accommodates the CC helix at its center . Descending from the top layer, the TRP loop embraces the LHD ring from the periphery, and continues into the stretcher helix. Below the LHD ring, intimately connected to the latter, the four stretcher helices converge into the central CC, like the stretchers of an umbrella into its shaft (pink and purple). Extensive intrasubunit interactions of the LHDs with TRP helix 1 and the pre-S1 helix (yellow) and PD  resembles that of other voltage-gated cation channels, and the VSLD is domain-swapped relative to the PD (blue boxes), and extensive hydrophobic interactions between the S4 and S5 helices of adjacent subunits (red). At the periphery of the top layer a tight interaction hub is formed between the short Pre-S1 helix (orange) and helices 12 and 13 of the LHD  are differently arranged, contain an extra β strand , and lack residues important for nucleotide binding, explaining insensitivity of TRPM2 to these nucleotides. Relative domain positioning is also significantly different in nvTRPM2 (Tetramer): when the pore domains of the three channels are superimposed, the displacement is 13–18 Å at the level of the NTDs (Subunit). Within the limitations of its lower resolution, the TRPM8 structure seems the least similar to TRPM4 and nvTRPM2, with a substantially different arrangement of its C-terminal helices relative to the transmembrane domain (TM-Stretcher-CC).Consistent with the high sequence conservation within the TRPM subfamily , the oveand β7b, , inset 3 nvTRPM2 . When vithe NTDs , Subunit+, the predominant cation in the buffer. E1037 in nvTRPM2 is replaced by a glutamine in hTRPM2, hTRPM4, and hTRPM5 (green box). This sequence difference might underlie the much larger Ca2+ preference of the nvTRPM2 pore. Indeed, the relative permeability for Ca2+ versus Na+ (pCa/pNa), estimated from the reversal potential of unitary currents under biionic conditions, is ~35 for nvTRPM2 (black), as opposed to ~0.45 for hTRPM2 , and helix S6. Asparagine 1017 in the turret loop is glycosylated, two N-acetyl-glucosamine molecules are clearly visible . The sel nvTRPM2 , black, ted from ), and ~0inset, red box), and additional negatively charged side chains (E1046 and E1050) line the upper parts of the vestibule (blue side chains). Crowding of four times four acidic residues in the tight external vestibule results in a larger negative surface charge density in nvTRPM2 compared to TRPM4 (pink boxes).The external vestibule of nvTRPM2 is lined by a double ring of negative charges , the sidestibule . It is aestibule , becauseestibule , stabilito TRPM4 . The shato TRPM4 : the nvTto TRPM4 , E1090 , as compared to ~25 pS for TRPM4 (red box), and one acidic residue in the internal vestibule. These differences likely account for its intermediate conductance  between TRPM4 and nvTRPM2. Indeed, inserting one or two negative charges into the external vestibule of hTRPM2 increased its conductance to ~72 pS, without much effecting relative Ca2+ permeability (2+ as the only permeating cation approached −10 pA at negative voltages (black symbols), which exceeds the throughput rate expected for a diffusion-limited channel , because its reintroduction abolished rundown , but replacing the native LDE triplet with a ‘human-like’ GY doublet results in clear rundown . Thus, the presence and proper positioning of D1041 and E1042 seems important for stabilization of the nvTRPM2 pore, but the E1042-K1047 salt bridge is not essential, because its disruption by mutations K1047A or K1047E does not result in rundown . Consis rundown . Possibl rundown . Because rundown  must refock cf., , below.As observed in other TRPM family channels, the gate of nvTRPM2 is formed by the S6 bundle crossing which constricts the pore to a diameter of less than 2 Å between residues I1082 and N1086 . Because2+ . The bound Ca2+ ion is coordinated by the side chains of residues E893 and Q896 of S2 and N918 and D921 of S3 in a pentacovalent geometry, E893 provides two coordinate bonds to the ion , and the geometry of Ca2+ coordination in nvTRPM2 resembles that seen in the Ca2+ bound structure of hTRPM4 , clear nterface , and is  the ion . TRP helof E1110  lines thof E1110 , facilitf hTRPM4 .2+ (left); a reduction of inward macroscopic current by millimolar cytosolic Ca2+ (right) reflects a reduction in unitary current amplitude due to pore block (2+ dependence of channel open probability (1/2 of ~2 μM (2+ coordination (2+ concentrations (left), reporting a dramatic loss of Ca2+ sensitivity. Correcting for the dose-dependent reduction in unitary current amplitude (2+ dependence of open probabilities (right). Estimated K1/2 for channel activation was ~40 μM for Q896A, whereas for N918A and D921A open probability failed to saturate even at 1250 μM (K1/2 >1 mM). The E1110A mutant displayed a more modest reduction in apparent Ca2+ affinity , consistent with the less direct involvement of the E1110 side chain in Ca2+ binding  channel currents that were stimulated only by 10–20% (2 addition (rightmost bars). Conversely, suppression of hTRPM2 activity upon PIP2 depletion was shown to be counteracted by raising cytosolic Ca2+  One possible explanation for these findings is that Ca2+ and PIP2 stabilize the open state independently of each other. Alternatively, the PIP2 headgroup itself might be directly involved in Ca2+ binding. Indeed, density for a phospholipid molecule is seen near the bound Ca2+ ion, in the immediate vicinity of the TRP box region of TRP helix 1 . The upper chamber, contained in the middle tier, is bounded from the top by the four TRP helices which act as ceiling joists that run parallel to the stretcher helices and converge into the cytoplasmic mouth of the pore (top and bottom). The empty upper chamber represents a relatively secluded volume of ~50000 A3, buried inside the protein . Indeed, the Ca2+ binding site can be viewed as a recess in a bent tunnel with a diameters > 4 Å (inset) that connects the upper chamber with the cytosol (left). Thus, in TRPM2, which is highly Ca2+ permeable, Ca2+ entering through the open pore can directly access the activating sites, without leaving the protein interior . In contrast, in monovalent-selective TRPM4 the central tunnel likely does not play such a role, as Ca2+ ions necessarily have to approach the channel from the cytosol. Interestingly, although both tunnels exist in TRPM4, their surfaces are positive (right).The cytosolic domains form a porous structure which houses a complex system of cavities crucial for channel function. The two ring-like structures formed in the bottom tier by the NTDs and ARDs and in the middle tier by the LHDs surround a common cytoplasmic cavity that extends in a direction perpendicular to the membrane surface . The fou protein , Video 1 protein , and fou protein . In addirs > 4 Å , inset t cytosol , Video 1 cytosol , left. Tpositive , right.2+ sites were shown to be inaccessible to extracellular Ca2+ in the closed-pore state, but saturated by extracellular Ca2+ entering through the pore in the open-channel state, leading to the suggestion that these sites are intracellular, but buried in some deep crevice, shielded from the protein surface, near the intracellular mouth of the pore (2+ binding sites in nvTRPM2 (2+ (2+ (gray bars) in the continuous presence of ADPR (black bar) caused nvTRPM2 currents to activate and deactivate, respectively, regardless of the applied membrane potential (colored bar and shading); the time constants of current decay in response to cytosolic Ca2+ removal (colored fit lines and numbers (in ms)) attested to a modest intrinsic voltage dependence of gating (green symbols). However, when extracellular [Ca2+] was 1 mM (2+ (black and gray bars) could be completely abolished by cytosolic Ca2+ removal only when the membrane potential was positive (gray shading), i.e., when the driving force for Ca2+ influx through the pore was small. Even at moderately positive voltages (+20 to+40 mV) the decay time constants upon cytosolic Ca2+ removal (red fit lines and numbers (in ms)) were greatly prolonged by the presence of external Ca2+ (purple symbols), approaching those measured in its absence (green symbols) only at extremely positive voltages (+60 to +80 mV) at which the driving force for Ca2+ influx is negligible. Such slowing of channel closure by external Ca2+ has been described for hTRPM2 , and subsided only when ADPR was also removed. One possible explanation is that at negative transmembrane voltages the number of Ca2+ ions accumulated in the cytoplasmic cavity during an open-pore event is too large to be completely dissipated during a subsequent closed event: the pore reopens before all Ca2+ ions would have left the cavity. This results in a low but substantial steady-state channel activity ) during which [Ca2+] in the upper chamber of each channel fluctuates between a very high (during open events) and a low but non-zero value (during closed events). Thus, whereas all four Ca2+ sites of a channel likely become saturated during each open event, some will lose Ca2+ during the subsequent closed event. Because channel opening rate is a property of the closed channel, the surviving current (yellow arrows) reflects channel opening rate under sub-saturating conditions, explaining the reduction in open probability upon removal of bulk cytosolic Ca2+.By contrast, at negative membrane potentials , the current that survives cytosolic Ca2+ removal might reflect channel cross-talk, that is, Ca2+ entering through a still-open pore re-opening a nearby closed channel, despite continuous rinsing of the cytosolic surface. Indeed, low millimolar concentrations of calcium buffer  were predicted to be inefficient for buffering cytosolic Ca2+ around a channel pore . Second, in addition to EGTA, our cytosolic solution contains 140 mM gluconate. Although gluconate is a low-affinity buffer, at 140 mM it binds ~88% of total cytosolic Ca2+ . Thus, the vectorial flushing effect is likely more important here than diffusion or the kinetics of Ca2+ binding to buffers – unless the shape of our patches is concave towards the bulk solution to an extent that severely limits access of the flowing bath solution to the patch surface. Fourth, the perhaps most compelling argument against Ca2+ accumulation at the patch surface being responsible for the residual currents in yellow arrows) is our finding that at negative voltages, in the presence of external Ca2+, nvTRPM2 currents activate, albeit with a delay, upon addition of cytosolic ADPR even in the absence of cytosolic Ca2+ (right): the magnitude of that ‘spontaneous’ current is identical to that which survives removal of pre-applied cytosolic Ca2+ (left). Because the spontaneous current activates from a state in which all pores in the patch have been closed for several seconds (section (e)), a duration more than sufficient to completely wash off even very high concentrations of pre-applied cytosolic Ca2+ ): upon the first such spontaneous opening of a channel its upper chamber is immediately flooded by Ca2+ which henceforth maintains that channel in an active state (section (g)).Alternatively, given the large density of channels in the patch ), to then remain activated by Ca2+ ions that enter through its pore and access the binding site through the central tunnel (section (c)). Indeed, extremely Ca2+ permeable nvTRPM2 does not even require an initiator spark: infrequent spontaneous openings of ADPR-bound channels allow for self-activation using exclusively extracellular Ca2+ (sections (f-g)) – that mechanism might be relevant even for moderately Ca2+ permeable hTRPM2 under the static conditions of a living cell. Because the ADPR-binding NUDT9H domain was not resolved here, further studies will be required to address the structural underpinnings of TRPM2 regulation by ADPR.In conclusion, we have presented here the cryo-EM structure of TRPM2 from an TRPM2 . Changes2 and 80% humidity.Insect cells were cultured at 28°C in sf-900 II SFM medium (GIBCO) supplemented with 5% FBS and 1% Antibiotic-Antimycotic. Mammalian cells were grown at 37°C in Freestyle 293 (GIBCO) supplemented with 2% FBS and 1% Antibiotic-Antimycotic. All cells were maintained with 8% CONematostella vectensis (nv) TRPM2 gene was synthesized into the pRML-13 BacMam expression vector (generous gift from Eric Gouaux) with a C-terminal GFP tag attached . The plasmid was transformed into DH10 Bac cells  to produce bacmid DNA, which was transfected into Sf9 cells  to generate recombinant baculoviruses. 10% (v/v) P3 virus was added to HEK 293S GnTI- cells  at 3 × 106 cells/ml. After 12 hr incubation at 37°C, protein expression was induced by 10 mM sodium butyrate at 30°C for 48 hr  supplemented with protease inhibitors (1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM benzamidine, 1 μg/ml aprotinin, 100 μg/ml trypsin inhibitor, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) and DNase (2 μg/ml). Membranes were solubilized with 1% 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG) and 0.1% cholesteryl hemisuccinate (CHS) at 4°C for 2 hr. After centrifugation at 75,000 g for 1 hr, the supernatant was mixed with GFP nanobody-coupled resin at 4°C for 2 hr. The resin was washed with 20 column volumes of Buffer A  to exchange LMNG and CHS with digitonin, and then incubated with PreScission protease (~10:1 w/w protein-to-enzyme ratio) at 4°C overnight to remove the C-terminal GFP tag. The GST-tagged protease was removed by binding to a glutathione-sepharose resin . The concentrated nvTRPM2 protein was further purified by gel filtration in Buffer A on a Superose 6 10/300 column .For protein purification, the cells were resuspended and homogenized in lysis buffer  and blotted using Vitrobot (FEI). Humidity was set to 100%, blotting time to 3 s and force to 0. After flash freezing in liquid ethane, the grids were stored in liquid nitrogen until screening and data collection.2. A total of 1619 images were collected. After manual inspection to remove poor quality images, 1550 images were used for further processing.The grids were initially screened on a 200 kV Talos Arctica (FEI) microscope, selected grids were then loaded into a 300 kV Titan Krios (FEI) microscope with a K2 summit detector (Gatan). Data were collected in super-resolution mode using Serial EM . Physicahttp://www.mrc-lmb.cam.ac.uk/kzhang), selected particles were manually inspected to remove false-positives and supplement false-negatives. Finally, a total number of 196,198 particles were input to RELION2  was not observed. Because the very N-terminal segments (6–23 and 41–51) had poor density and were disconnected from the rest of the protein, their registers are somewhat uncertain and they were built as poly-alanines.The transmembrane domain (TMD) was modeled using SWISS-MODEL  based onThe refinement was performed in Phenix  with sechttp://www.pymol.org), Chimera, HOLE  and stored at 18°C in a frog Ringer's solution supplemented with 1.8 mM CaCl2 plus 50 μg/ml gentamycin sulfate (Sigma).Ovarian lobes were removed from anaesthetized TRPM2 gene was synthesized into the pGEMHE expression vector . Mutations were introduced using Stratagene QuikChange. cDNA was transcribed in vitro using T7 polymerase (mMESSAGE mMACHINE T7 kit Thermo Scientific), and cRNA stored at −80°C. Xenopus laevis oocytes were injected (Drummond Nanoject) with 0.1–10 ng of WT or mutant nvTRPM2 cRNA, and recordings were done 1–3 days after injection.The nv2+-activated chloride currents, as described earlier for hTRPM2 ; 1 mM Na-EGTA or 8 mM Ca(gluconate)2 was added to obtain free [Ca2+] of ~1 nM , and either 1 mM EGTA (to obtain ‘zero’ (~1 nM) Ca2+), or 20 μM to 10 mM Ca-gluconate2 (to obtain 3 to 1250 μM free [Ca2+]). For unitary current measurements under biionic conditions 2). Na2-ADPR (Sigma), Dioctanoyl-PIP2 , and poly-L-lysine (Sigma) were added to the bath solution from 200 mM, 2.5 mM, and 15 mg/ml aqueous stock solutions, respectively. The continuously flowing bath solution was exchanged using computer-driven electronic valves; solution exchange time constant was <100 ms. Macroscopic and unitary nvTRPM2 currents were recorded at the indicated membrane potentials , digitized at 10 kHz , and filtered at 2 kHz.Macroscopic and unitary nvTRPM2 currents were recorded in excised inside-out patches at 25°C in symmetrical 140 mM Na-gluconate based solutions to avoid activation of endogenous Car hTRPM2 . The tipof ~1 nM  or ~1 mMof ~1 nM  and 6. Tnditions  the pipei)-voltage (V) relationship under biionic conditions (blue curve), but not for nvTRPM2. Thus, for nvTRPM2 the i-V plot was fitted to a quadratic function to estimate the reversal potential. Because for nvTRPM2 unitary currents could not be resolved at membrane potentials >+ 4 mV, the extrapolated reversal potential (~+28 mV) was also verified in, and found roughly consistent with, macroscopic recordings.For recordings under asymmetrical ionic conditions  liquid jnditions , was wel2+, or by 125 μM Ca2+ + 25 μM dioctanoyl-PIP2, were calculated by dividing steady current in the test segment (I) with that in 125 μM Ca2+ (I125) in the same patch. Open probabilities (Po) normalized to that in 125 μM Ca2+ , were calculated as Po/Po;125=(I/I125)/(i/i125) /(I125+PIP2/I125) and Po/Po;125+PIP2=/. For D921A nvTRPM2 channel currents in the absence of PIP2 were too small for reliable cursor measurement. Thus, for this mutant i and N⋅Po in 13, 125, or 1250 μM cytosolic Ca2+ was estimated using dwell-time analysis, and fractional Po under such conditions  calculated as Po/Po;125+PIP2=(i⋅N⋅Po)/(I125+PIP2).Fractional current activation in 100 μM ADPR by test concentrations of cytosolic Ca Ca2+see ). To emp2+, current decay time courses upon cytosolic Ca2+ removal  segments of recording, from ≥3  patches.Cryo-EM density map of nvTRPM2 has been deposited in the electron microscopy data bank (EMDB) under accession code EMD-7542. Atomic coordinates of nvTRPM2 have been deposited in the protein data bank (PDB) under accession code: 6CO7. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.2+ explains unique gating regulation" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom, Kenton J Swartz is a member of our Board of Reviewing Editors and the evaluation has been overseen by Kenton Swartz as the Reviewing Editor and Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Leon D. Islas (Reviewer #2); Alexander Sobolevsky (Reviewer #3).Thank you for submitting your article "Structure of a TRPM2 channel in complex with CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2. Although PIP2 is not resolved definitively and the structure was solved in the absence of calcium and ADP-ribose, a calcium binding site near the internal ends of S2 and S3 appears to be identified that is similar to that recently reported for the TRPM4 channel. The external vestibule appears wide enough to pass ions and contains many acidic residues that are important for ion permeation, and the internal pore appears to be closed. One of the more fascinating aspects of the structure is the presence of upper and lower chambers within the cytosolic domain, as well as the tunnels connecting the upper chamber to the putative calcium binding sites and the lower chamber to the bulk solution. The authors show us why the nvTRPM2 channel does not inactivate (unlike the human channel), they show that residues coordinating calcium have either altered calcium affinity or are non-functional, and they present an intriguing phenomenon whereby calcium permeation from outside to inside can reach the calcium binding sites even when the internal surface of the protein is being perfused by calcium buffers. Overall, we think this is one of the nicest TRP channel structures to be reported recently and the work is highly appropriate for eLife.This is a wonderful manuscript reporting the first near-atomic resolution structure of the TRPM2 channel and a detailed mechanistic study to investigate several of the most intriguing new structural features. The TRPM2 channel is activated by intracellular calcium, ADP-ribose and PIPEssential revisions:2+ binding site” they use the word trace. Also, the composition of the solution used to prepare grids is not given. Is it buffer A? We couldn't find the composition of that solution anywhere? It would be nice to see larger density maps in this region with stronger justification. Do you see density for calcium in the half maps? Do you see stronger density for the acidic residues you think are binding calcium compared to other regions of the protein where acidic residues are not involved in hydrogen bonds or ion binding (and are therefore more damaged by the electron beam)? In TRPM4, the authors compared maps with and without calcium to make a stronger case. We are not requesting any new experiments, just expand the presentation/justification and perhaps tone down the conclusion about a calcium ion being present in the maps and models.1) We are not sure how definitive the authors can be about identification of calcium bound to its putative site. The extensive mutagenesis and comparison with TRPM4 makes us believe that identification of the site is correct, but not whether a calcium ion is actually bound. In subsection “Structure determination” the authors state that the structure was solved in the presence of calcium, but in subsection “Ca2) One of the reviewers thinks that it is almost certain that you have calcium ion accumulation on the internal face of you patches for the experiments shown in Figure 5 and Figure 6. Many of the currents are several nA and these are inside out patches where the membrane will adopt a concave shape towards the bulk solution. This doesn't negate the conclusion that calcium is coming from the outside, but it’s not surprising that calcium can't be washed away at negative voltages when one considers the geometry of the patch and the kinetics of calcium binding to buffers. Please see Stern, 1992, a paper that describes a model that predicts your results for any calcium permeable channel being studied in inside-out patches. According to that model, it might be possible to rule out calcium accumulation using high concentration of a fast buffer such as BAPTA, but you would probably also have to reduce your expression level considerably. You should deal with this point as you see fit, but it would be a shame to misinterpret your results if they have nothing to do with the really beautiful chambers and tunnels.2+, since one would expect that the entering Ca2+ would saturate the binding sites if accessing it through the tunnels. Perhaps intracellular Ca2+ induces a separate conformation that allows more efficient coupling to the activation gate?3) Another reviewer asked why the reactivating current is not as high as that obtained with intracellularly applied Ca4) nvTRPM2 represents a highly calcium-permeable TRP channel (PCa/PNa~35). Zhang et al. focus on nvTRPM2 structural features that distinguish its calcium permeability from other TRPMs. However, a brief discussion of how specific these structural features compared to other calcium-permeable channels would broaden the scope of this manuscript and make it more interesting to the general reader. For example, how similar to or different from are the structural mechanisms of high calcium permeability in TRPM2 versus most highly calcium-selective TRP channels TRPV5/6? Essential revisions:2+ binding site” they use the word trace. Also, the composition of the solution used to prepare grids is not given. Is it buffer A? We couldn't find the composition of that solution anywhere?1) We are not sure how definitive the authors can be about identification of calcium bound to its putative site. The extensive mutagenesis and comparison with TRPM4 makes us believe that identification of the site is correct, but not whether a calcium ion is actually bound. In subsection “Structure determination” the authors state that the structure was solved in the presence of calcium, but in subsection “Ca2+, so we now state "trace amounts of Ca2+" in subsection “Ca2+ binding site”. Free Ca2+ concentration in a typical saline without added Ca2+ or Ca2+ buffers is in the μM range , the same buffer as that used for gel filtration. We now explicitly state that the EM sample was concentrated from the "gel filtration peak fractions in Buffer A" . The buffer was not supplemented with Ca2+, as people have measured (unpublished). Therefore, given the apparent K1/2 of ~2 µM for nvTRPM2 , it is not surprising that bound Ca2+ ions are present in our EM map.2003) we have measured 13 µM), and the standard Vitrobot filter paper we used for freezing grids also contains a certain amount of CaIt would be nice to see larger density maps in this region with stronger justification. Do you see density for calcium in the half maps? Do you see stronger density for the acidic residues you think are binding calcium compared to other regions of the protein where acidic residues are not involved in hydrogen bonds or ion binding (and are therefore more damaged by the electron beam)? In TRPM4, the authors compared maps with and without calcium to make a stronger case. We are not requesting any new experiments, just expand the presentation/justification and perhaps tone down the conclusion about a calcium ion being present in the maps and models.2+ binding site . In the full map and both half maps, the Ca2+ densities are all very clear, and the densities for the acidic amino acids which participate in Ca2+ binding (D921 and E893) are indeed much stronger than those of adjacent residues that are not involved in hydrogen bonding or Ca2+ coordination .To allow better judgement, we now show larger density maps  for the region around the Ca2) One of the reviewers thinks that it is almost certain that you have calcium ion accumulation on the internal face of you patches for the experiments shown in Figure 5 and Figure 6. Many of the currents are several nA and these are inside out patches where the membrane will adopt a concave shape towards the bulk solution. This doesn't negate the conclusion that calcium is coming from the outside, but it’s not surprising that calcium can't be washed away at negative voltages when one considers the geometry of the patch and the kinetics of calcium binding to buffers. Please see Stern, 1992, a paper that describes a model that predicts your results for any calcium permeable channel being studied in inside-out patches. According to that model, it might be possible to rule out calcium accumulation using high concentration of a fast buffer such as BAPTA, but you would probably also have to reduce your expression level considerably. You should deal with this point as you see fit, but it would be a shame to misinterpret your results if they have nothing to do with the really beautiful chambers and tunnels.+, K+, NMDG+) per BAPTA molecule (at pH=7.1) into the bath solution would drastically alter unitary conductance by causing either reversal potential shift or pore block. Thus, we acknowledge that we cannot fully exclude the possibility of some Ca2+ accumulation at the cytosolic patch surface, but we also provide four arguments which make this unlikely (subsection “Cytoplasmic cavities and tunnels and access to the Ca2+ binding site”):This is a valid concern that we are fully aware of. We did observe the phenomenon depicted in Figure 5E and Figure 6 even in patches with much smaller currents . Unfortunately, high concentrations of BAPTA, as suggested in the Stern paper, cannot be tested here, because the inevitable introduction of four cations , when the extracellular solution contains 140 mM Na+ and 1 mM Ca2+ , the majority (~74%) of inward nvTRPM2 currents are carried by Na+ ions (which account for ~85% of entering cations).First, based on the unitary conductances measured with Na2+ , and its high concentration precludes its saturation.Second, in addition to EGTA, our cytosolic solution contains 140 mM gluconate. Although gluconate is a low-affinity buffer, at 140 mM it binds ~88% of total cytosolic Ca2+ binding to the buffer, and partly by the rate of diffusion of the buffer ligand, because the buffer molecules in the vicinity of the pore become rapidly saturated and must be replaced by unbound buffer molecules via diffusion. Here the cytosolic face of the patch is immersed into a rapidly flowing solution. Considering the flow rate of ~1 cm/s of our bath solution and the ~10 nm diameter of a channel protein , the entire solution volume in contact with the surface of a channel is displaced by fresh Ca2+-free solution every ~1 μs, during which time ~2 Ca2+ ions enter through its pore (assuming a Ca2+ current of 0.6 pA per open channel). Thus, the vectorial flushing effect is likely more important here than diffusion or the kinetics of Ca2+ binding to buffers – unless the shape of our patches is concave towards the bulk solution to an extent that severely limits access of the flowing bath solution to the patch surface.Third, Stern's calculation applies to a static environment (like the cytosol of an intact cell during whole-cell recording) in which buffering around the pore is limited partly by the kinetics of Ca2+ accumulation at the patch surface being responsible for the residual currents in Figure 5E, (yellow arrows) is our finding that at negative voltages, in the presence of external Ca2+, nvTRPM2 currents activate, albeit with a delay, upon addition of cytosolic ADPR even in the absence of cytosolic Ca2+ : the magnitude of that "spontaneous" current is identical to that which survives removal of pre-applied cytosolic Ca2+ . Because the spontaneous current activates from a state in which all pores in the patch have been closed for several seconds ), a duration more than sufficient to completely wash off even very high concentrations of pre-applied cytosolic Ca2+ , it cannot be explained by bulk Ca2+ accumulation. Rather, its delayed activation must reflect a very low "spontaneous" channel opening rate of ADPR-bound channels in the absence of bound Ca2+ ): upon the first such spontaneous opening of a channel its upper chamber is immediately flooded by Ca2+ which henceforth maintains that channel in an active state ).Fourth, the perhaps most compelling argument against Ca2+, since one would expect that the entering Ca2+ would saturate the binding sites if accessing it through the tunnels. Perhaps intracellular Ca2+ induces a separate conformation that allows more efficient coupling to the activation gate?3) Another reviewer asked why the reactivating current is not as high as that obtained with intracellularly applied Ca2+ entering only through the pore but cytosolic bulk Ca2+ concentration kept close to zero, Ca2+ concentration in the upper chamber of each channel fluctuates between a very high (during open events) and a low but non-zero value (during closed events). This is because once the gate has closed, Ca2+ ions start to dissipate from the chamber through the various windows and tunnels. Thus, whereas all four Ca2+ sites of a channel likely become saturated during each open event, some will lose Ca2+ during the subsequent closed event. Because channel opening rate is a property of the closed channel, the current that survives cytosolic Ca2+ removal , or the spontaneous reactivating current , reflects channel opening rate under sub-saturating conditions. This explains the lower open probability compared to that seen in the presence of saturating cytosolic Ca2+. We have added this explanation to the text (subsection “Cytoplasmic cavities and tunnels and access to the Ca2+ binding site”)During "spontaneous" channel activity, with Ca4) nvTRPM2 represents a highly calcium-permeable TRP channel (PCa/PNa~35). Zhang et al. focus on nvTRPM2 structural features that distinguish its calcium permeability from other TRPMs. However, a brief discussion of how specific these structural features compared to other calcium-permeable channels would broaden the scope of this manuscript and make it more interesting to the general reader. For example, how similar to or different from are the structural mechanisms of high calcium permeability in TRPM2 versus most highly calcium-selective TRP channels TRPV5/6?2+ throughput rate of the nvTRPM2 pore exceeds that expected for a diffusion-limited channel . This is a clear indication that local Ca2+ concentration in the nvTRPM2 outer vestibule is far higher than that in the bulk solution, suggesting that the negative charges in the nvTRPM2 outer vestibule also act as "recruitment sites".This is an interesting point which we now address in the text (subsection “Ion channel pore”). Although sequence conservation between nvTRPM2 and TRPV6 in the filter and outer vestibule region is low, for both channels the outermost filter position is formed by a ring of four carboxylates , and additional 3-4 negatively charged side chains per subunit are present in the external vestibule. In TRPV6 the latter were termed "recruitment sites" . Indeed, closer inspection of nvTRPM2 unitary current sizes  indicates that the Ca


This interaction facilitates slow Ca2+-dependent inactivation after Orai1 channel activation by wild-type STIM1 or a constitutively active STIM1 mutant. We define the CaM-binding site in STIM1, which is adjacent to the STIM1–Orai1 coupling region. The binding of Ca2+-CaM to activated STIM1 disrupts the STIM1–Orai1 complex and also disassembles STIM1 oligomer. Based on these results we propose a model for the calcium-bound CaM-regulated deactivation of SOCE.Store-operated calcium entry (SOCE) is a major pathway for calcium ions influx into cells and has a critical role in various cell functions. Here we demonstrate that calcium-bound calmodulin  is elicited by the interaction of oligomerized STIM1 with plasma membrane Orai channels. Here, the authors show that this process is regulated by calmodulin, which binds STIM1 and disassembles the STIM1-Orai1 complex and STIM1 oligomers. Store-operated calcium entry (SOCE) is the major extracellular Ca2+ influx process in excitable and, particularly, in non-excitable cells3. Previous studies have identified the molecular constituents of SOCE: an endoplasmic reticulum (ER) Ca2+ sensor, the stromal interaction molecule (STIM)6 and a plasma membrane (PM) pore-forming subunit, Orai9. By sensing Ca2+ depletion within the ER, STIM undergoes oligomerization and translocates to ER–PM junctions, where it activates highly Ca2+-selective Orai channels10. Influx of Ca2+ through Orai channels provides precise Ca2+ signals that are essential for regulating long-term responses such as gene expression and cell growth11.As an intracellular second messenger, Ca2+ entry through Orai channels modulates their activity through a process called Ca2+-dependent inactivation (CDI), which is an important negative feedback mechanism that maintains intracellular Ca2+ homeostasis. CDI consists of fast CDI (FCDI) and slow CDI (SCDI), which have distinct kinetics and sites of action12. FCDI occurs within tens of milliseconds after channel activation and is controlled by Ca2+ binding to a site located several nanometers from the pore14. The FCDI process was regulated by various factors, including the STIM1-to-Orai1 expression ratio15, the intracellular loop of Orai1 and the conserved negatively charged cluster in the STIM1 COOH terminus18. SCDI occurs within tens of seconds of channel activation and is driven by a global rather than local Ca2+ rise20. The SOCE-associated regulatory factor (SARAF) has been reported to facilitate SCDI of SOCE21 through interaction with the STIM1 C-terminal inhibitory domain (448–530)22. Interestingly, SARAF was not able to regulate SCDI after Orai1 channel activation by the constitutively active STIM1 mutant D76A22. Moreover, SARAF-dependent SCDI appears to be regulated by multiple factors, such as Caveolin, E-Syt1, Septin4 and PIP223.Ca2+-binding sites. CaM adopts a closed conformation in the Ca2+-free state (apo-CaM) and an open conformation in the Ca2+-bound state (holo-CaM)24. Both states are able to bind various target proteins and consequently transduce different Ca2+ signals25. Ion channels are major targets of CaM regulation. For example, the CDI of voltage-gated Ca2+ channels regulated by CaM is well established26. Apo-CaM is pre-associated with the IQ domain, whereas Ca2+ influx induces subtle conformational rearrangements between CaM and the Ca2+-inactivation region, which facilitates CDI. For Orai channels, one previous study has shown that CaM might be the Ca2+ sensor for FCDI28 but the results of a more recent report have contradicted this argument29.Calmodulin (CaM) consists of an N-lobe and a C-lobe, each of which contains two Ca31. However, the deactivation mechanism is largely unknown. In this study, we demonstrated that CaM associates with the core region of STIM1 in a Ca2+-dependent manner. Consequently, calcium-bound CaM disrupts the STIM1–Orai1 complex and disassembles STIM1 oligomers, thereby inducing deactivation of the SOCE.After more than 30 years of intensive studies, the activation mechanism of SOCE has been well documented2+11. Its C-terminal region is located in the cytosolic portion of the protein and contains the core domain SOAR/CAD/OASF/Ccb9, which associates with the Orai1 channel35 . To investigate whether CaM is able to interact with the cytosolic C-terminal regions of STIM1 molecules, we designed various human STIM1 constructs and transiently transfected HEK293T cells. CaM-Sepharose beads were used for a pull-down experiment. We found that two constructs (SOAR and STIM1-c2) interacted with CaM in a Ca2+-dependent manner. Neither STIM1-c1, which perturbed SOAR formation nor STIM1-c3, STIM1-c4 and STIM1-c5, which contain residues after SOAR, associated with CaM .Human STIM1 is a type I membrane protein that contains 685 amino acids. Its N-terminal region is located inside the ER and contains an EF-SAM domain, which senses CaCaM Fig. , thus su2+, CaM interacted with SOAR with high affinity (Kd = 0.23 µM). In the absence of Ca2+, CaM did not bind to SOAR. As a control, we also used CaM mutants (CaM-4EF) in which glutamate was mutated to glutamine to diminish Ca2+ loading at each Ca2+-binding site36. The titration of CaM-4EF into SOAR in the presence of Ca2+ showed no detectable binding signal  experiments were carried out. Our results showed that in the presence of Canal Fig. . These r2+ but not in its absence  increases between SOAR-YFP and CaM-CFP but not between SOAR-YFP and the mutant CaM-4EF-CFP  and fluorescence resonance energy transfer (FRET) experiments to verify that the interaction between SOAR and CaM occurs in cells. When coexpressed with myc-SOAR, CaM-YFP was able to pull down SOAR in the presence of Cance Fig. . Furthernce Fig.  and, mornce Fig.  after ceCFP Fig. . These r2+-dependent interaction between CaM and SOAR, we used electrophysiology to determine whether CaM participates in the regulation of Orai1 SCDI. Our results showed that including purified CaM in the pipette solution increased the extent of the SCDI of Orai1 current that was induced by SOAR and the extent of inactivation in a CaM concentration-dependent manner  spectroscopy. Multiple human STIM1 constructs were screened, and one (STIM1 363–416) produced well-resolved peaks in the NMR 1H-15N HSQC spectrum. Each residue was assigned unambiguously  construct occurred with a slow-to-intermediate exchange rate, and that many cross-peaks were broadened with the addition of Ca2+-CaM 41. In contrast, titrating Ca2+-CaM into an 15N-labeled STIM1 (363–416) mutant, L390S-F391S, resulted in a faster exchange rate  and the STIM1 (363–416) L390S-F391S mutant  docking with Ca2+-CaM 43 and SOAR (PDB code: 3TEQ)30. Residues L390 and F391 were buried deeply inside the C-terminal lobe of Ca2+-CaM  interacting with Ca2+-CaM by using CaM-Sepharose bead pull-down and co-IP experiments. Our results showed that the SOAR mutant L390S-F391S (LF-SS) eliminated the interaction of SOAR with Ca2+-CaM -coupled multiangle light scattering experiment showed that the molecular weights of WT SOAR and LF-SS mutant proteins were similar and corresponded to the size of a dimer  for Ca15N-STIM1 and 15N-STIM1-L390S-F391S produced similar 15N-HSQC spectra  into HEK293T cells and measured the FRET Eapp values. We observed robust FRET signals between Orai1-CFP and the WT SOAR-YFP (SOAR-WT) at the PM. In contrast, the FRET signals between Orai1-CFP and the SOAR-YFP mutant (SOAR–LF-SS) were negligible , which is the site where SOAR (and STIM1) binds to Orai1ble Fig. . Moreoveble Fig. , thus inble Fig. . To testlux Fig. . These d2+-CaM binding to SOAR, we tested whether Ca2+-CaM could directly disrupt the SOAR–Orai1 complex in vitro. GFP-Orai1 and myc-SOAR were co-transfected into HEK293T cells. The SOAR–Orai1 complex was purified by GFP-Trap and washed with purified CaM in the absence or presence of Ca2+. Our results showed that CaM-containing solution largely disrupted the SOAR–Orai1 complex in a Ca2+-dependent manner, compared with CaM-free solution  between the resting and activation states when it was co-expressed with mCherry-Orai1. In the resting state, both the STIM1-WT and STIM1 mutant (STIM1–LF-SS) were distributed across the ER, whereas Orai1 was uniformly distributed on the PM  alone or with mCherry-Orai1. In cells expressing STIM1 alone, the WT and mutant were distributed across the ER in resting cells  into HEK293T cells. In the resting state, the STIM1 WT and mutant STIM-LF-SS showed similar FRET Eapp values. These values were higher than the control values, thus indicating that the mutation of residues L390 and F391 did not impair STIM1 dimer formation  was able to pull-down both overexpressed CaM and endogenous CaM after cells were stimulated by thapsigargin, thus providing good evidence that Ca2+-CaM has the capability to associate with exposed SOAR in vivo. Moreover, using a FRET technique, we observed that extracellular Ca2+ influx induced CaM access to SOAR. The control CaM-4EF mutant was not able to access SOAR. These data suggest that after extracellular Ca2+ influx causes cytosolic Ca2+ elevation, CaM senses and loads with Ca2+ and then accesses and binds to SOAR in activated STIM1.CaM has a crucial role in maintaining Ca53 and found that the region around L390-F391 represents the most likely binding site. We further located the specific binding site through NMR titration and validated this binding site through immunoprecipitation methods. So far, two regions in STIMs were found to interact with Ca2+-CaM55. One region is located in the STIM1 C-terminus and the other is located in the STIM2 SOAR domain. First, the very C-terminal polybasic region (667–685) in STIM1 was found through ITC and NMR methods to interact with Ca2+-CaM. This region (667–685) did not contain any known CaM-binding site and consists of several basic and hydrophobic residues. ITC experiments in which the polybasic peptide was titrated into CaM revealed a strong binding affinity between these two molecules (1 μM)54. Second, through pull-down and SPR methods, the region 459–482 in SOAR of STIM2, which corresponds with residues 368–391 in SOAR of STIM1, was found to interact with Ca2+-CaM55. This region is also rich in basic and hydrophobic residues and did not contain any known CaM-binding motif. Therefore, CaM seems to bind with STIM1 in an unconventional way.Interestingly, the SOAR region does not contain a known CaM-binding motif. We then searched for the CaM-binding site in SOAR in a CaM target database2+-CaM. These residues are adjacent to the Orai1-binding site of SOAR, thus suggesting that Ca2+-CaM binding to SOAR will interfere with the SOAR–Orai1 complex or the STIM1–Orai1 complex. Furthermore, the affinity value that we measured between SOAR and Ca2+-CaM was ~0.2 μM, ~1,000-fold higher than the affinity (~200 μM) of SOAR for the Orai1 channel, according to previous reports56. This large difference in affinity allows Ca2+-CaM to compete with Orai1 for association with SOAR, thus leading to the breakdown of the SOAR–Orai1 complex or the STIM1–Orai1 complex.SOAR is the basic unit of STIM1 that binds and activates the Orai1 channel. It has been demonstrated that the polybasic SOAR residues 384–387 are crucial for SOAR binding to Orai1. Interestingly, we identified residues L390 and F391 as necessary for SOAR binding to Ca2+-CaM binding site of SOAR or STIM1, leads to the failure of puncta formation and activation. Thus, we infer that Ca2+-CaM binding to the activated STIM1 oligomer may lead to a shift in equilibrium from an STIM1 oligomer to a less aggregated form. In our system that uses purified CaM, we indeed observed that the STIM1 oligomer became less aggregated after the addition of Ca2+-CaM. This result was further confirmed by FRET measurements in cells.Puncta formation represents STIM1 aggregation and activation. Our results showed that the mutation of residues Leu390 and Phe391, which are part of the Ca2+ levels, Ca2+-CaM disrupts the SOAR–Orai1 complex or the STIM1–Orai1 complex, resulting in the inactivation of the Orai1 channel. This process was consistent with the SCDI of the Orai1 channel. Indeed, including purified CaM in the pipette facilitated Orai1 SCDI. More importantly, when the Orai1 channel was activated by the constitutively active STIM1 mutant D76A, CaM was also able to facilitate SCDI. When inhibitors were used to inhibit endogenous CaM, the SCDI of the Orai1 current was reduced. Thus, Ca2+-CaM induced SCDI appears to differ from SARAF-induced SCDI. It has been reported that SARAF-induced SCDI may be mediated by the interaction of SARAF with residues 448–530 of STIM1, which are located after SOAR22. In addition, STIM1 and SARAF are localized in the ER and interact in the basal state with filled Ca2+ stores23, suggesting that SARAF functions by holding STIM1 in a resting state. This statement is consistent with data that SARAF does not inactivate the Orai1 channel if the constitutively active STIM1 mutant D76A or 4E-4A is used22. Therefore, we speculate that Ca2+-CaM has a dominant role in disrupting the STIM1–Orai1 complex and disaggregating the STIM1 oligomer. The inactive STIM1 conformation is presumably maintained by SARAF to avoid the spontaneous activation of STIM1. More evidence is required to test this possibility in the near future.The Orai1 channel is characteristic of both FCDI and SCDI. FCDI occurs within milliseconds, whereas SCDI occurs within minutes. Our results showed that after CaM responds to elevated cytosolic Ca2+-CaM  according to the manufacturer’s instructions.HEK293T (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Hyclone). HEK stable expressing Orai1-CFP  were cultured in DMEM supplemented with 10% fetal bovine serum and 100 μg mlBglII and EcoRI sites to generate STIM1-CFP, STIM1-GFP and STIM1-YFP. The sequence of SOAR was amplified by PCR and cloned into pCMV-Myc (Clontech) vector between EcoRI and BglII sites to generate the Myc-SOAR. Full-length cDNA of human Orai1 was inserted into pmCherry-C1 vector (Clontech) between BglII and EcoRI sites to generate mCherry-Orai1. Full-length cDNA of human Orai1 was inserted into pCMV-Myc vector between EcoRI and BglII sites to generate Myc-Orai1. GFP-STIM1 truncations were generated by introducing stop codon at the specific position. Full-length cDNA of rat CaM was cloned into pET-33(b) vector (Novagen) between NcoI and XhoI. mCherry-Orai1, SOAR-GFP, myc-Orai1, myc-SOAR and MBP-SOAR have been previously described30. Mutations were generated by using the Quick-change mutagenesis kit (Agilent). Primers used in this study were listed in Supplementary Table Full-length complementary DNA of human STIM1 was inserted into pECFP-C1, pEGFP-C1 and pEYFP-C1 vectors (Clontech) between Escherichia coli. The cells were collected and suspended in lysis buffer  and lysed by sonication. After centrifugation at 15,000 g for 1 h, the supernatant was loaded onto a MBP-Trap HP column  equilibrated with equilibration buffer . The target protein was eluted using elution buffer  and cleaved with Tobacco Etch Virus protease overnight at 4 °C. The protein was applied to a HiTrap SP column  equilibrated with 20 mM MES pH 6.0, 50 mM NaCl, 1 mM dithiothreitol (DTT) and 1 mM EGTA and eluted by increasing the NaCl concentration. Target protein was collected and further purified by using a Superdex 200 10/300 GL column . Protein concentrations were determined using a BCA assay kit (Pierce). The purification of the LF-SS mutant followed the same protocol for the SOAR WT.Human SOAR WT and LF-SS mutant were expressed in E. coli. The cells were collected and suspended in lysis buffer  and lysed by sonication. For the WT CaM, after centrifugation at 15,000 g for 1 h, the supernatant was supplemented with NaCl and CaCl2 to a final concentration of 500 mM NaCl and 5 mM CaCl2. The supernatant was loaded onto a HiPrep Phenyl FF (High sub) 16/10 column  equilibrated with equilibration buffer . Protein was eluted using elution buffer  and further purified by using a HiLoad 26/600 Superdex 200 column . For CaM-4EF, the supernatant was applied to a HiPrep Q FF 16/10 column  equilibrated with 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT and 1 mM EGTA. Protein was eluted by increasing the NaCl concentration. Target protein was collected and further purified by using a HiLoad 26/600 Superdex 200 column . Protein concentrations were determined using a BCA assay kit (Pierce).CaM and CaM-4EF were expressed in E. coli. The cells were collected and suspended in lysis buffer  and lysed by sonication. After centrifugation at 15,000 g for 1 h, the supernatant was loaded onto an Ni-NTA affinity column and washed extensively with lysis buffer. The target protein was eluted using elution buffer  and cleaved with PreScission protease overnight at 4 °C. The protein was applied to a HiTrap SP column  equilibrated with 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT and 1 mM EGTA, and eluted by increasing the NaCl concentration. Target protein was collected and further purified by using a Superdex Peptide 10/300 GL column . Protein concentrations were determined using a BCA assay kit (Pierce). Mutants were expressed and purified by following the protocol for STIM1 (363–416).The STIM1(363–416) construct was expressed in 15N- or 13C-labeled protein, protein was expressed and purified by following the same protocol used for the unlabeled protein, whereas the Lysogeny broth (LB) medium was replaced with M9 medium supplemented with 15N-NH4Cl or 13C-glucose.For the 2 or 1 mM EGTA. Cell lysates were centrifuged at 15,000 g for 30 min at 4 °C. The supernatant was quantified with a BCA assay kit (Pierce). One milligram of cell lysate (1 mg ml−1) was incubated with 30 μl prewashed CAM-Sepharose 4B beads  for 2 h in 4 °C. The beads were washed six times with lysis buffer and eluted in 30 μl gel-loading buffer by heating at 95 °C for 10 min. Then, 10 μl of the elution was loaded onto 12–15% SDS-polyacrylamide gel electrophoresis (PAGE) gels or 10–16% tricine–SDS-PAGE 63 and transferred to a polyvinylidene difluoride (PVDF) membrane for western blot analysis.Transfected cells were washed twice with Tris-buffered saline (TBS) and lysed in chilled buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail (Roche) and 2 mM CaCl2 or 1 mM EGTA. For thapsigargin treatment, cells were treated with 1 μM thapsigargin before lysis for 10 min. Cell lysates were centrifuged at 15,000 g for 30 min at 4 °C. The supernatant was quantified with a BCA assay kit (Pierce). Next, 0.5 mg cell lysate (1 mg ml−1) was incubated with rabbit c-Myc-agarose  or GFP-Trap  for 2–4 h in 4 °C. The beads were washed three times with lysis buffer and eluted in 40 μl gel-loading buffer by heating at 95 °C for 10 min. Then, 15 μl of the eluate was loaded onto 8–12% SDS-PAGE or 10–16% tricine–SDS-PAGE gels and transferred to a PVDF membrane for western analysis. Proteins were immunoblotted using anti-myc , anti-GFP  and anti-tubulin . The protein–antibody complexes were detected by chemiluminescence. Uncropped original scans of blots are shown in Supplementary Fig. Transfected cells were washed with TBS twice and lysed in chilled buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, protease inhibitor cocktail (Roche) and 2 mM CaClCells were plated on glass-bottom dishes and transfected at 50% confluence. Cells were imaged in Ringer’s solution. Thapsigargin  was used for store depletion. Enhanced CFP (ECFP), enhanced GFP (EGFP), enhanced YFP (EYFP) and mCherry were excited at 458, 488, 514 and 594 nm, respectively. Fluorescence emission was collected at 465–505 nm for ECFP, 500–540 nm for EGFP, 520–560 nm for EYFP and 605–650 nm for mCherry. All images were captured at room temperature with a × 63 and 1.40 numerical aperture oil-immersion objective lens controlled by LAS software on a Leica SP8 confocal microscope.l-lysine (Sigma-Aldrich) and transfected at 60% confluence. Transfected cells were loaded with 2 μM Fura-2 AM (Invitrogen) in Ringer solution at room temperature for 30 min. After loading, cells were transferred to Fura-2 AM-free solution for 30 min. The Ringer solution contained (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 10 d-glucose and 20 HEPES-NaOH, pH 7.4. Fluorescence imaging was undertaken at room temperature using a Leica DMI6000B microscope with a × 63 and 1.40 numerical aperture oil-immersion objective lens controlled by LAS software. Consecutive excitation occurred at 340 and 380 nm every 2 s, and emission was collected at 510 nm. Intracellular Ca2+ concentration is shown as the 340/380 ratio. Data are shown as the mean ± SEM.HEK293T was plated on glass-bottom dishes coated with poly-64. Briefly, bleed-through and direct excitation containment were removed according to the equation Fc = IDA − aIAA − dIDD (assuming b = c = 0). The bleed-through factor “d” was determined for cells expressing ECFP alone . The direct excitation factor “a” was determined for cells expressing EYFP alone . The apparent FRET efficiency (Eapp) was calculated by the equation Eapp = Fc/(Fc + GIDD), where Eapp represents the fraction of donor (ECFP) exhibiting FRET. The microscope-specific constant G was determined by using the D1ER cameleon .HEK293T or HEK Orai1-CFP were plated on glass-bottom dishes and transfected with ECFP (donor)- and EYFP (acceptor)-tagged constructs at 50% confluence. Imaging was performed at room temperature using a Leica DMI6000B microscope equipped with high-speed fluorescence-external filter wheels for a CFP-YFP index-based FRET experiment. Images were collected every 10 s using a ×63 oil objective  and processed using the Biosensor Processing Software 2.1 package in MATLAB R2014a. FRET was determined by pixel-by-pixel calculation using apparent FRET efficiency calculation methods2 at pH 6.5 unless otherwise indicated. The NMR data were processed with Topspin 2.1 and chemical shifts were assigned with the Sparky and Cara packages. The protein backbone assignments were completed with 3D CBCANH and CBCA(CO)NH spectra.All NMR spectra were recorded at 298 K with a Bruker Avance 600 MHz NMR spectrometer equipped with a QCI-cryoprobe. Protein samples were in 20 mM MES and 5 mM CaCl15N-HSQC spectra. The binding experiment was performed with 15N-HSQC spectra of 15N-STIM1 titrated by CaM. Cross-peak intensity of STIM1 was compared in the presence or absence of CaM. The chemical shift perturbations of STIM1 were plotted with CaM and the dissociation constant (Kd) was simulated with Origin.The interaction between CaM and STIM1 was analyzed by 2. After trypsin digestion, transfected cells were plated onto 35 mm dishes with culture medium for at least 1 h to attach cells for electrophysiology. Experiments were performed at room temperature by using the tight-seal whole-cell configuration with the following voltage protocol: 50 ms voltage step to −100 mV from a holding potential of 0 mV, followed by a voltage ramp increasing from −100 to + 100 mV in 50 ms with a frequency of 0.5 Hz. To measure the SCDI, we used a pipette solution with 135 mM Cs aspartate, 8 mM MgCl2, 1.2 mM EGTA and 10 mM Cs-HEPES with CaM or its mutant or inhibitors (pH 7.2). The extracellular 0 Ca2+ solutions contained the following: 130 mM NaCl, 4.5 mM KCl, 22 mM MgCl2, 10 mM TEA-Cl, 10 mM d-glucose and 5 mM Na-HEPES (pH 7.4). The extracellular Ca2+-containing solutions contained the following: 130 mM NaCl, 4.5 mM KCl, 20 mM MgCl2, 2 mM CaCl2, 10 mM TEA-Cl, 10 mM d-glucose and 5 mM Na-HEPES (pH 7.4). After whole-cell configuration in the 0 Ca2+ extracellular solution, the first whole-cell currents that were recorded were denoted leak currents. Then, the 0 Ca2+ extracellular solution was exchanged slowly with 2 mM Ca2+ extracellular solution via a perfusion system to record Orai1 currents. Whole-cell currents were amplified with an Axopatch 700B and digitized with a Digidata 1550A system (Molecular Devices). All currents were low-pass filtered at 2 kHz and sampled at 10 kHz. pCLAMP software (Molecular Devices) was used for data acquisition and analysis. To reflect the SCDI process, the current was normalized as follows: the peak current density (pA pF−1) was set as −1.0 and the current densities during the measurement were displayed as the ratio of the peak current density to get normalized current densities and were plotted as a function of the recording time. Data points represent the mean ± SEM.HEK293 cells were transiently transfected with CFP-Orai1 and SOAR-YFP, STIM1-YFP or the STIM1-YFP mutant in a ratio of 1:2 for 12–24 h at 37 °C with 5% CO2 or 1 mM EGTA. For the STIM1–Orai1 and STIM1–STIM1 complexes, 3 μM ionomycin was used to induce Ca2+ store depletion before lysis. Cell lysates were centrifuged at 15,000 g for 30 min at 4 °C. The complex was pooled with 40 μl GFP-Trap (ChromoTek) for 2 h in 4 °C. After samples were washed twice with lysis buffer, 0.3 mM Ca2+ + CaM or EGTA + CaM was added to the complex for binding for 4 h in 4 °C. The beads were washed three times with lysis buffer and eluted in 40 μl gel-loading buffer by heating at 95 °C for 10 min. Then, 10 μl of the elution was loaded onto 7–15% SDS-PAGE gels or 10–16% tricine–SDS-PAGE (for low molecular-weight protein)63 and transferred to a PVDF membrane for western blot analysis.Transfected cells were washed twice with TBS and lysed in chilled buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, protease inhibitor cocktail (Roche) and 2 mM CaCl2 or 1 mM EGTA. To determine the binding affinity of SOAR and CaM, 500 μM CaM or CaM-4EF was injected into 50 μM SOAR. Data were analyzed in the Microcal Origin software.Measurements were performed at 16 °C using an ITC-200 microcalorimeter . Samples were buffered with 25 mM Tris-HCl (pH 7.5) containing 200 mM NaCl and 2 mM CaClProtein secondary structure was recorded in a MOS-450 CD spectrometer (BioLogic) equipped with a temperature controller. Spectral data were measurement at 22 °C using a 1 mm path length quartz cell with 10 μM protein between wavelength of 190–260 nm and using buffer for blank correction.−1. Protein concentration was calculated from the absorbance at 280 nm and the light scattering data were collected at 663 nm. Molecular weight was calculated using the ASTRA software.Multi-angle light scattering was performed in-line with SEC using a DAWN HELLOS α instrument equipped with an Optilab Refractive Index Detector (Wyatt Technology). Protein (100 μM) was injected into a Superdex 200 10/300 column  equilibrated with 25 mM Tris-HCl pH 7.5, 200 mM NaCl at a flow rate of 0.5 ml mint-test with SPSS (IBM) software. No specific randomization or blinding protocols were used. Results are expressed as the mean ± SEM of at least three independent experiments. Differences with a P-value of < 0.05 were considered significant: *P < 0.05, **P < 0.01 and ***P < 0.001.Statistical significance was determined using unpaired Student’s The authors declare that the data supporting the findings of this study can be found within the paper and its Supplementary informationPeer review file


The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10–25 ms, reached 50% Fmax with ∼1200 effectively absorbed photons and saturated (0ΔF/F ∼ 10–20) with 10000–30000 photons. In Ca2+ free bath, smaller (0ΔF/F ∼4), long latency (∼200 ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Any tiny Ca2+ free rise remaining without exchanger activity was equivalent to <10 nM (0ΔF/F ∼0.1), and unlikely to play any role in phototransduction. However, using the high affinity ratiometric indicator INDO-1, this Ca2+ free signal was reported to be unaffected in null mutants of the only InsP3 receptor (IP3R) gene in the Drosophila genome 2+ free rise measured using the genetically encoded indicator GCaMP6f was substantially reduced following RNAi knockdown of the IP3R and proposed that InsP3-induced Ca2+ release played a critical role in phototransduction.Measurements using fluorescent CaninaE) promoter. We performed measurements in dissociated ommatidia allowing control of extracellular solutions, and also in vivo from completely intact flies by imaging the rhabdomeres in the “deep pseudopupil” (DPP) 2+ signals in vivo in response to physiologically relevant stimuli. We paid particular attention to the origin of the Ca2+ rise under Ca2+ free conditions, and found that it was unaffected in IP3R mutants, but strictly dependent upon both Na+ influx and Na+/Ca2+ exchanger activity. We conclude that any light-induced release from internal stores is minimal (<10 nM), slow, and unlikely to play any direct role in phototransduction.In the present study, we generated flies expressing GCaMP6f 22.1Drosophila melanogaster) were reared on standard medium  from 0ΔF/F values, we assumed our Fmax value of 23.5, the published Kd value (290 nM), and Hill slope (n = 2.7) Maximum and minimum fluorescence of GCaMP6f 33.12+ in Drosophila photoreceptors we expressed GCaMP6f directly under the control of the Rh1 opsin (ninaE) promoter (see methods), thereby driving expression specifically in the major photoreceptor class (R1-6). Whole-cell recordings from photoreceptors of these flies (ninaE-GCaMP6f) showed that their basic light responses were indistinguishable from wild-type , the fluorescence rapidly rose to 0ΔF/F values >10 within ∼200 ms. Although the signals were broadly similar, responses recorded from the DPP were faster than those recorded in dissociated ommatidia, probably because the DPP samples fluorescence predominantly from the rhabdomeres, whilst the ommatidium signal is dominated by cytosolic GCaMP6f. Apart from the pedestal (0F) and maxF values, these signals per se are relatively uninformative: firstly because the blue excitation light is a super-saturating, non physiological stimulus, and secondly because GCaMP6f, with a Kd of 290 nM in vitro2+ concentrations in excess of ∼1–2 μM, whilst [Ca2+] in the photoreceptor is believed to reach values close to 1 mM in the rhabdomere and 10–50 μM in the cell body In order to monitor Caild-type . Imagingild-type , Fig. 5Aild-type  B [24]. 2+ rise with respect to physiologically relevant intensities we used 2-pulse protocols in which brief (1–2 ms) calibrated flashes of green light were delivered 300 ms before measuring fluorescence  and saturating at intensities of 10000–30000 photons when measured in vivo from the DPP . The small difference in sensitivity may again reflect the predominantly rhabdomeric (DPP) vs cytosolic (dissociated ommatidium) source of the signals; but may also reflect the influence of the dissociation procedure, axial (DPP) as opposed to side-on (ommatidia) illumination, and/or the very different methods used to calibrate intensity; namely counting quantum bumps in whole-cell recordings from dissociated ommatidia, as opposed to measuring the metarhodopsin to rhodopsin photoisomerisation rate spectrophotometrically for the DPP (see methods 2.3).In order to measure the intensity dependence of the Carescence . The ped the DPP  D. F/log343trp null mutant flies lacking the dominant and more Ca2+ permeable of the two light-sensitive channels. Although maximum 0ΔF/F values to saturating illumination were similar to wild-type , F/log I functions determined with 2-pulse protocols were ∼10–20 x less sensitive, with 50% maxF obtained with brief flashes containing 14800 ± 3100 (n = 8) effective photons  flashes whilst varying the delay of the blue excitation light. Depending on intensity, Ca2+ rises were observed with latencies of ∼10–25 ms, peaking in 200–300 ms and then returning to baseline over a period of 2–4 s with a half time (t½) of ∼900 ms with dimmer test flashes  there is a smaller and slower rise in Ca2+, the source and role of which is controversial ninaE-GCaMP6f flies, this “Ca2+ free” signal can be recorded with excellent signal-to-noise ratio and reached oΔF/F values of ∼1-6 (mean ∼3.2 ± 0.3 n = 24) after 2 s, which is now well within the dynamic range of GCaMP6f. Importantly, there was no detectable rise until at least ∼200 ms after light onset  would not be reliably resolved  calibrated flashes 2 s before the blue excitation. Now, approximately 40000 effectively absorbed photons (>1 per microvillus) were required to elicit a 50% rise, which is ∼20–40 x more than for responses in physiological (Ca2+ containing) solutions or in vivo . The source of this is uncertain P24norpA null PLC mutants  Ca2+ stores would seem the most obvious explanation for the Ca2+ free signal; however, we found that it was completely unaffected in null mutants (mosaics) of the only InsP3 receptor gene (itpr) in the Drosophila genomes  than our standard Ca2+ free solution (1 mM EGTA). We therefore also repeated measurements using 0.5 mM EGTA both in puffer pipettes and also after whole bath perfusion, but again in every case (n = 17 ommatidia in 6 flies) only slow (∼200 ms latency) responses were observed. Very occasionally (<5% of more than 100 ommatidia) we did see a more rapid Ca2+ signal; however, it was immediately clear that this was due to failure to adequately perfuse the ommatidium with Ca2+ free solution (e.g. a blocked puffer pipette). We can only speculate that a similar explanation may account for the rapid signals reported by Kohn et al. Light-induced release from internal  or an essentially non-permeant cation (NMDG+).In an early study using the ratiometric indicator INDO-1 we reported that extracellular Naiminated . This wa+ for a substantial Ca2+ free rise might reflect re-equilibration of Na+/Ca2+ exchange following the massive Na+ influx associated with these stimuli + gradient, and not Na+ influx, was necessary and suggested some other Na+ dependent process was required for release from internal stores. To test the requirement for Na+ influx, as opposed to a Na+ gradient we expressed GCaMP6f in trpl;trp double null mutants lacking all light-sensitive channels + and Ca2+ influx, irrespective of the extracellular solution. Now, even in the presence of normal external Na+ and Ca2+, the GCaMP6f signal was at least as severely reduced as in wild-type ommatidia bathed in Ca2+ and Na+ free solutions, leaving again a tiny slow rise to a maximum oΔF/F of <0.1 after 1–2 s  dark adaptation, these measurements yielded similar results, with at most a tiny rise similar to that recorded in dissociated ommatidia ; however, following perfusion with La3+  in the same ommatidia, only a tiny slow rise similar to that seen in Ca2+ and Na+ free solutions, or in trpl;trp double mutants remained  would reflect a Ca2+ rise of the order of ∼10 nM. This signal developed slowly, was only detectable with very bright stimuli, was unaffected by the IP3R null mutation, and because of its tiny size one cannot even be confident it represents a Ca2+ signal.In summary, these results indicate that Na3.42+ free rise is due to re-equilibration of Na+/Ca2+ exchange following Na+ influx calx gene). We therefore expressed GCaMP6f in the severe calx hypomorph, 1calx, which has no detectable exchanger activity 2+ free solution we were no longer able to detect any light-induced increase in GCaMP6f fluorescence in 1calx mutants beyond a tiny residual signal similar to that seen in Ca2+ and Na+ free solutions in wild-type backgrounds (2+ rise in vivo (DPP) or from dissociated ommatidia in normal bath (i.e. 1.5 mM Ca2+) in 1calx flies was broadly similar to that in wild-type flies; however, reflecting the pivotal role of the exchanger in Ca2+ extrusion, recovery to baseline in the dark was much slower, particularly following brighter flashes (in vivo 2-pulse F/log I functions) was also approximately 2–3 x lower than in wild-type , 1calx mutants showed an attenuated LIC, presumably due to the inhibitory effects of Ca2+. However, in Ca2+ free solutions, responses in 1calx were actually significantly larger than in wild-type  than in otherwise wild-type flies. The accelerated Ca2+ free rise in ninaE-calx flies was still dependent upon Na+ influx because it was no longer seen after perfusion with Ca2+ and Na+ free solutions (Na+ substituted for Cs+), leaving instead a tiny residual rise similar to that in wild-type Ca2+ and Na+ controls , over-expression of the exchanger in ninaE-calx flies had the predictable and opposite effect of suppressing Ca2+ rises in the F/log I function and also accelerating recovery to baseline  dependent upon Na+ influx strongly suggests that the rise can be attributed to the activity of the exchanger in response to massive Na+ influx, as originally proposed In summary, our results show that the Ca42+ signals in Drosophila photoreceptors were first studied over 20 years ago using Ca2+ indicator dyes 2+ indicators ninaE) promoter ensuring exclusive expression in R1-6 photoreceptors with wild-type morphology and physiology. The excellent signal-to-noise ratio of recordings in ninaE-GCaMP6f flies was distinctly superior to that in GMR-Gal4/UAS-GCaMP6f flies, and in many cases the maximum 0ΔF/F ratio approached or exceeded 20  or in dissociated ommatidia bathed in physiological solutions, the GCaMP6f signal reached 50% Fmax at intensities equivalent to ∼1000–2500 effectively absorbed photons. It is believed that the elementary single photon response (quantum bump) is generated by activation of Ca2+ permeable channels (TRP and TRPL) within a single microvillus and that the consequent Ca2+ rise in the affected microvillus reaches near mM levels d 290 nM, saturating at 1–2 μM), to a first approximation the 0ΔF/F values under physiological conditions are probably best interpreted as the proportion of microvilli “flooded” with Ca2+. In total, the rhabdomere contains ∼30000 microvilli, meaning that 50% Fmax is reached when only ∼3–8% of the microvilli have been activated by a photon. This implies that the Ca2+ influx into a single microvillus must spread to at least the immediately neighbouring microvilli within the timeframe of the response. In ninaE-calx flies over-expressing the Na+/Ca2+ exchanger, or in trp mutants lacking the major Ca2+ permeable channel, 50% maxF was only obtained with flashes containing ∼ 12000-15000 effective photons, which should activate ∼50% of the microvilli, suggesting that in these flies Ca2+ is largely prevented from spreading to neighbouring microvilli under the same conditions.2+ concentration in dissociated ommatidia  assuming in vitro calibration data 0F measured in Ca2+ free solution in the same ommatidia, the mean dark-adapted value in normal bath was 0.77 ± 0.14 (mean ± S.E.M. n = 11), which would be equivalent to ∼80 nM  and higher in 1calx mutants .The dark-adapted “pedestal” level can be used to gain an estimate of the resting Ca2+ levels during response recovery, although the initial decrease  will still be subject to saturation effects. With relatively dim flashes (up to ∼1000 effectively absorbed photons) the GCaMP6f signal in wild-type backgrounds fell to near baseline within ∼2–3 s with a half time (t ½) of ∼1 s , and slowed in 1calx mutants .The recovery of GCaMP6f fluorescence to baseline is likely to be a reasonably accurate reflection of the falling Ca of ∼1 s . This is4.22+ free bath fall within the dynamic range of GCaMP6f and allow estimates of the absolute Ca2+ levels reached under these conditions (e.g. 0ΔF/F of 6 corresponds to ∼200 nM). We used these signals to investigate the long disputed origin of the light-induced rise in cytosolic Ca2+ in Ca2+ free solutions. Originally, using INDO-1, we found that this Ca2+ free rise was dependent upon extracellular Na+ and suggested that the rise might be due to re-equilibration of Na+/Ca2+ exchange in response to the massive light-induced Na+ influx that persists under these conditions + for a significant Ca2+ rise in Ca2+ free solutions, Na2+, but reported that a rise still occurred in Ca2+ free bath in the presence of Na+ when the photoreceptors were voltage clamped at the Na+ equilibrium potential to prevent Na+ influx. They concluded that a Na+ gradient − but not influx − was required, that the Ca2+ free rise reflected release from internal stores, and that the requirement of extracellular Na+ reflected involvement of some other Na+ dependent process, such as Na/H transport. But how this might affect release of Ca2+ from intracellular stores is far from clear. A more recent study from the same lab reported that the Ca2+ free rise was attenuated following RNAi knockdown of the IP3R 3R mosaic eyes The smaller signals recorded in Ca2+ free signal further. We first confirmed that it was all but abolished in the absence of external Na+, whether substituted for Li+, Cs+, K+ or NMDG+. Importantly, we found that the rise was also effectively eliminated in trpl;trp double mutants both in vivo and in dissociated ommatidia despite the presence of normal extracellular solutions containing both Na+ and Ca2+. Although it might be argued that, for some reason, PLC activity (and hence InsP3 generation) was compromised in trpl;trp mutants, convincing evidence indicates that net PLC activity is in fact greatly enhanced in trpl;trp due to the lack of Ca2+ and PKC dependent inhibition of PLC. Thus the rate and intensity dependence of PIP2 hydrolysis, measured using GFP-tagged PIP2 binding probes are greatly enhanced in trpl;trp mutants + influx is indeed required for the Ca2+ free rise. Crucially, the involvement of the Na+/Ca2+ exchanger in this rise was confirmed by finding that it was essentially eliminated in an exchanger mutant , but greatly accelerated in ninaE-calx photoreceptors over-expressing the exchanger  in cells perfused with EGTA buffered solutions, when free Ca2+ in the bath should be reduced to low nM levels. We do not have an unequivocal answer to this, and assuming the standard equation for the Na+/Ca2+ exchange equilibriumThe question remains, how Na+/Ca2+ exchange to raise Ca2+ into the range we observed. However, at least three, not mutually exclusive factors might result in higher cytosolic Cai levels than predicted. Firstly, external Ca2+ might be relatively resistant to buffering in the intra-ommatidial space, and specifically the extremely narrow spaces between the microvilli or their bases, where the exchanger is believed to be localised o remaining, Eq. i would be reached with 70 mM Nai, 110 mM Nao and the cell depolarised to 0 mV . Although one might also expect Ca2+ influx via the light-sensitive channels at such Cao concentrations, experiments buffering external Ca2+ at different concentrations with EGTA showed that direct Ca2+ influx signals could only be detected once external Ca2+ was raised above ∼400 nM . Secondly, resting cytosolic Ca2+ concentration is determined not only by the exchanger, but also by any other Ca2+ fluxes, which might include tonic leakage from intracellular compartments such as endoplasmic reticulum (ER) or mitochondria. Massive Na+ influx would compromise the ability of the exchanger to counter any such fluxes. A third possibility is that, contrary to conventional dogma, the exchanger might also be expressed on intracellular membranes of endoplasmic reticulum or other Ca2+ containing compartments and that Na+ influx leads to re-equilibration of Na+/Ca2+ exchange across these.it would seem difficult for reverse Na2+ rise in Ca2+ free bath is strictly dependent upon both Na+ influx and the activity level of the Na+/Ca2+ exchanger, but unaffected in null IP3R mutants. Its time-course, with no detectable rise for ∼200 ms, also appears much too slow to play any role in initiating the light response, which has a latency of ∼10 ms and peaks within ∼100–200 ms in response to bright illumination even under Ca2+ free conditions [34][e.g. 34]. The residual GCaMP6f signal remaining in the absence of Na+ influx and/or in the absence of Na+/Ca2+ exchanger activity − whether achieved by Na+ substitution, trpl;trp or calx mutants − was also still observed in IP3R mutants and was so small that it is questionable whether it reflects a Ca2+ signal. Because of the rapid inhibition of PLC by Ca2+ influx under physiological conditions 2+ release under physiological conditions would be even less. Together with a study in which we found no phototransduction defects in null IP3R mutants , these results suggest that InsP3-induced Ca2+ release plays no significant role in Drosophila phototransduction.Whatever the exact mechanism, our results indicate that the CaThe authors declare no competing financial interests2+ imaging experiments. SA performed and analysed Ca2+ imaging and whole-cell electrophysiology experiments. C-HL performed molecular biology and fly genetics. SA and C-HL commented on the MS.RCH conceived and designed the research, wrote the paper and performed and analysed Ca


Isolated rat hearts were perfused with Krebs–Henseleit solution (Control), followed by Ca2+-depletion, and then Ca2+-repletion after Ca2+-depletion (Ca2+-paradox) by Langendorff method. During heart perfusion left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), rate of pressure development (+ dP/dt), and pressure decay (-dP/dt) were registered. Control LVDP (127.4 ± 6.1 mmHg) was reduced during Ca2+-depletion (9.8 ± 1.3 mmHg) and Ca2+-paradox (12.9 ± 1.3 mmHg) with similar decline in +dP/dt and –dP/dt. LVEDP was increased in both Ca2+-depletion and Ca2+-paradox. Compared to Control, myofibrillar Ca2+-stimulated ATPase activity was decreased in the Ca2+-depletion group (12.08 ± 0.57 vs. 8.13 ± 0.19 µmol Pi/mg protein/h), besides unvarying Mg2+ ATPase activity, while upon Ca2+-paradox myofibrillar Ca2+-stimulated ATPase activity was decreased (12.08 ± 0.57 vs. 8.40 ± 0.22 µmol Pi/mg protein/h), but Mg2+ ATPase activity was increased (3.20 ± 0.25 vs. 7.21 ± 0.36 µmol Pi/mg protein/h). In force measurements of isolated cardiomyocytes at saturating [Ca2+], Ca2+-depleted cells had lower rate constant of force redevelopment  and unchanged active tension, while those in Ca2+-paradox produced lower active tension (12.12 ± 3.19 kN/m2) and ktr,max (3.21 ± 23) than cells of Control group . In biochemical assays, α-myosin heavy chain and cardiac troponin T presented progressive degradation during Ca2+-depletion and Ca2+-paradox. Our results suggest that contractile impairment in Ca2+-paradox partially resides in deranged sarcomeric function and compromised myofibrillar ATPase activity as a result of myofilament protein degradation, such as α-myosin heavy chain and cardiac troponin T. Impaired relaxation seen in Ca2+-paradoxical hearts is apparently not related to titin, rather explained by the altered myofibrillar ATPase activity.This study aimed to explore the potential contribution of myofibrils to contractile dysfunction in Ca This memorable, but unexpected observation was first reported by Zimmerman and Hülsmann five decades ago  is a key element of I/R injury  units, and accordingly the pCa of relaxing solution was 9.0, whereas the pCa of maximal activating solution was 4.75. Protease inhibitors were added to all solutions freshly: phenylmethylsulfonyl fluoride (PMSF): 0.5 mM; leupeptin: 40 μM; and E-64: 10 μM. All chemicals were purchased from Sigma-Aldrich, Corp. .Contractile function of skinned ventricular cardiomyocytes , respectively. During Ca2+-contractions, a so-called release–restretch maneuver, i.e., slack test, was applied in order to estimate the rate constant of force redevelopment (ktr). Actin–myosin turnover constant ktr was determined at pCa values ranging from 4.75 to 6.0. Note that force redevelopment could not be fitted accurately at pCa > 6.0 due to the low signal-to-noise ratio. Plots indicating active force values at each pCa normalized to the corresponding maximum at pCa 4.75 as 1.0 were fitted by a specific sigmoidal function in Origin 6.0 analysis program . It follows that the pCa value for the half-maximal contraction indicated by pCa50 defines per se the Ca2+-sensitivity of the contractile machinery. The steepness of the Ca2+-sensitivity curve, reflecting the myofilament cooperation, was calculated by a modified Hill equation and expressed as a coefficient (nHill). Ca2+-independent passive force of the examined cardiac cell was measured by the shortening to 80% of initial preparation length in relaxing solution. Original forces (in µN) recorded by this experimental composition were adapted by cross section area—in µm2 calculating from the width and height—of each individual cell indicating cardiomyocyte tension (expressed in kN/m2).Maximal and submaximal Ca2, 1.0 EGTA, 1.0 EDTA, 0.5 DTT and CaCl2, 0.3 Calpain Inhibitor I , leupeptin, and Phosphatase Inhibitor Cocktail 1 , pH 7.5. Protein concentration was determined according to the Lowry method. Homogenates were aliquoted and immediately boiled in SDS sample buffer for 3 min. To ascertain protein degradation in the crude homogenates, one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed  in a non-stacking gradient gel system  followed by silver staining  might differ in the myofibrils and in the cytosol. Based on this theory, one could speculate that depletion of ATP in the myofilament compartment might lead to impaired relaxation  by activating ATPases, and consequently internal Ca2+-overload promotes ATP depletion and accelerates rigor tension development  is relatively low during Ca2+-paradox [2+-depleted and Ca2+-paradoxical hearts, global impairment of systolic function was associated with depressed myofibrillar Ca2+-stimulated ATPase activity. It has been known for a long time that myosin ATPase determines the velocity of muscle contraction [2+-activated contraction rate  are tightly correlated with Ca2+-stimulated ATPase activity of myofibrils as elementary for initiating power stroke [2+-stimulated ATPase activity is associated with reduced Ca2+-dependent contraction, resulting in systolic dysfunction, like in Ca2+-paradox hearts.An increased diastolic tension and pressure therefore raise the lack of ATP as required for relaxation. Cytosolic ATP content, however, is not only preserved during hypercontracture that is provoked by intracellular Ca-paradox . Neverth-paradox . Accordilaxation , therebyelopment . In myofkinetics . Experimactivity . Indeed,n ATPase . These rtachment . Since mtachment , the relelopment , 35. Acc-paradox . On the traction , and thur stroke . Consequ2+-paradox, and described a significant decrease in maximal active tension and ktr,max of still surviving isolated cardiomyocytes after Langendorff-perfusion. These results from single-cell preparations are consistent with our LV contractility findings, since we consider reduced active tension as the cellular basis of depressed LVDP. Similar drop in +dP/dt and −dP/dt is the global reflection of reduced ktr,max by reason of depressed Ca2+-stimulated ATPase activity. In contrast to Ca2+-dependent parameters, passive tension of isolated cardiomyocytes was not affected at all during Ca2+-depletion then Ca2+-replacement. Thus, passive myofilament components of diastole appear unlikely to be primarily responsible for myocardial stiffening in Ca2+-paradox.Beyond that, for the first time we made an attempt to test cellular mechanical function in Ca2+-paradox. Based on previous observations, limited degradation of myofibrils might occur after Ca2+-paradox, suggesting cTnT release through membrane destruction [2+-overload exhibited degradation of cTnT. A potential cross-linking between troponin subunits or their fragments and other cardiac proteins was therefore suggested in cell death after Ca2+-paradox [2+-depletion and Ca2+-repletion, although we did not observe cross-linked cTnT forms.Here we detected a gradual damage of essential contractile proteins, such as α-MHC and cTnT en route to Catruction . Likewis-paradox . These r2+-paradox demonstrate impaired global and cellular contractility that is accompanied by decreased Ca2+-stimulated ATPase activity potentially as a result of myofilament protein degradation. According to data provided in this study, we conclude that significant deterioration in cardiac relaxation seen in Ca2+-paradoxical hearts is probably due to a failure in cross-bridge cycling because of an altered myofibrillar ATPase activity, and is apparently not directly related to titin. On the other hand, it is likely that one of the molecular bases of dramatic decrease in systolic function in Ca2+-paradox might be α-MHC and cTnT cleavage, resulting in a collective reduction of interdependent parameters of Ca2+-dependent force generation at the level of cardiomyocytes, myofibrils, and isolated heart.In summary, isolated rat hearts suffered Ca


ANKRD26 gene. Patients have thrombocytopenia and increased risk of myeloid malignancies, in particular, acute myeloid leukemia (AML). Given the association of variants in the ANKRD26 5’UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5’UTR. We found variants in four patients. One patient had the c.−125T>G substitution in the 5’UTR, while three patients carried two different variants in the 5’ end of the ANKRD26 coding region (c.3G>A or c.105C>G). Review of medical history showed that the patient carrying the c.−125T>G was actually affected by typical but unrecognized THC2, highlighting that some apparently sporadic AML cases represent the evolution of a well-characterized familial predisposition disorder. As regards the c.3G>A and the c.105C>G, we found that both variants result in the synthesis of N-terminal truncated ANKRD26 isoforms, which are stable and functional in cells, in particular, have a strong ability to activate the MAPK/ERK signaling pathway. Moreover, investigation of one patient with the c.3G>A showed that mutation was associated with strong ANKRD26 overexpression in vivo, which is the proposed mechanism for predisposition to AML in THC2 patients. These data provide evidence that N-terminal ANKRD26 truncating mutations play a potential pathogenetic role in AML. Recognition of AML patients with germline ANKRD26 pathogenetic variants is mandatory for selection of donors for bone marrow transplantation.Thrombocytopenia 2 (THC2) is an inherited disorder caused by monoallelic single nucleotide substitutions in the 5’UTR of the The online version of this article (doi:10.1186/s13045-016-0382-y) contains supplementary material, which is available to authorized users. ANKRD26 gene [ANKRD26 in hematopoiesis is poorly understood. A recent investigation indicated that thrombocytopenia of THC2 patients is caused by ANKRD26 overexpression in megakaryocytes due to defective downregulation by RUNX1 and FLI1, which, in turn, derives from impaired binding of these transcription factors to the mutated 5’UTR [Thrombocytopenia 2  is an autosomal dominant disorder caused by monoallelic single nucleotide substitutions in the 5’UTR of the D26 gene . The roled 5’UTR .ANKRD26 5’UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5’UTR. Genomic DNA was obtained from peripheral blood at the time of diagnosis.A growing body of evidence indicates that a significant proportion of apparently sporadic, adult-onset AML cases originate from a germline predisposition, which often is not recognized , 6. GiveWe found three different variants in four patients, whose clinical features are reported in Additional file ANKRD26 that is predicted to cause the loss of the physiologic start codon (p.Met1?). In both patients, we could analyze the DNA from different tissues , which demonstrated the germinal origin of the variant. Finally, one patient had the c.105C>G substitution resulting in the generation of a stop codon at position 35 (p.Tyr35*). We thus investigated the effects of these two variants in the 5’ end of the ANKRD26 coding region.Two patients carried the c.3G>A variant of ANKRD26 mRNA expression was strongly increased (about ninefold changes) in the patient compared with healthy controls , and the SDI antibody recognizing an ANKRD26 internal epitope (residues 289–388)  or the mutant To investigate the stability of the mutant proteins in cells, we blocked the protein synthesis by adding cycloheximide to HeLa cultures 24 h after transfection and measured the kinetics of the subsequent reduction of the amounts of transfected proteins. These experiments showed that both variants presented a similar stability as the WT protein  transplantation from a family donor, in order to avoid the use of HSC from a donor affected by the same inherited disorder. In fact, several reports indicate that the use of HSC from donors with germline mutations predisposing to hematological malignancies resulted in the development of donor-derived leukemia in the recipient and/or poor transplant engraftment [In summary, the analysis of a large case series showed that variants in the raftment –10. Of nANKRD26 coding sequence resulting in the truncation of the protein N-terminus also have a regulatory effect, causing ANKRD26 overexpression and thus playing a potential role in AML. In fact, ANKRD26 overexpression is the proposed pathogenetic mechanism for both thrombocytopenia and predisposition to AML in THC2 patients [ANKRD26 overexpression though a mechanism independent of RUNX1/FLI1 interaction with the 5’UTR of the gene and possibly due to increased mRNA stability. In this way, the transcription factors are still able to bind the 5’UTR and downregulate ANKRD26 in megakaryocytes, thus avoiding thrombocytopenia. Whatever the mechanisms of ANKRD26 upregulation, we showed that these N-truncated isoforms are stable in cells and have a strong ability to activate the MAPK/ERK pathway. Although further investigation is required, the present data strongly suggest that N-terminal truncating mutations of ANKRD26 have a potential pathogenetic role in apparently sporadic AML. Since our investigation was restricted to non-familial AML cases, prevalence of ANKRD26 pathogenetic variants in AML could be greater than we found.Moreover, we observed that mutations in the patients . Since n