Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits  reside in the intermembrane space. Here, we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.Ca The MCUne space . MICU1 ccoveries  suggest 2+ to enter the matrix, Ca2+ must first permeate the outer mitochondrial membrane (OMM) through the largely open VDAC (voltage-dependent ‘anion’ channel), a beta-barrel channel into the intermembrane space (IMS). From the IMS, Ca2+ crosses the nearly impermeant inner mitochondrial membrane (IMM) in a highly regulated manner into the mitochondrial matrix through the small conductance, highly selective MCUcx  mouse embryonic fibroblasts (MEFs)  . DRP1 is complex . Importachondria . We alsochondria . MCU, EM2+]i dependence of the MCUcx current in mitoplasts from WT DRP1-KO MEFs, and shows the absence of Ca2+ current (ICa) in MCU-KO or EMRE-KO. Additionally, it shows an important feature of the MCUcx; in the absence of extramitochondrial Ca2+ (control trace), there is outward current at positive potentials resulting from the efflux of Na+ through the MCUcx due to the 110 mM Na+ gluconate in the matrix from the patch pipette , an MCU-mediated Na+ current (INa) is observed, and this current also depends on the presence of MCU and EMRE ( pipette . When exrmeation  and no oll lines . From thll lines . We showand EMRE .2+]i increase (elicited by SERCA inhibitor thapsigargin) was followed, after a short delay, by [Ca2+]m elevation as detected by a genetically-encoded Ca2+ indicator Cepia targeted to mitochondria  below which there is no MCUcx-medicated Ca2+ influx. 2+. This threshold is largely gone in MICU1-KO cells , an observation that is inconsistent with the plug hypothesis  was significantly reduced in MICU1-KO , as was eduction . Therefocx, we used a novel test to characterize the MCUcx channel properties independent of Ca2+ conductance. We used Na+ current via MCUcx (INa) in the absence of both Ca2+ and Mg2+ (using EDTA)  , to calitoplasts . This prtoplasts . Also, ton to WT . Importatoplasts . From th+/+Dnm1l). In these cells, the amplitudes of ICa and INa were the same as in -/-Dnm1l MEFs  achieved by our fast application system . Under these conditions, no outward ICa was observed either before or after [Ca2+]i elevation in either WT or MICU1-KO. However, as expected, 1 mM [Ca2+]i induced a robust inward ICa  IMM patches (cx current (iCa via a single MCUcx channel) is very small if measured in physiological [Ca2+]i, it must be recorded at high [Ca2+]i = 105 mM to enable proper resolution  of the MCUcx is increased by the hyperpolarization of the IMM as was also shown previously (cx as well as sub-conductance openings at ~80% and ~60% of the amplitude of the fully open iCa (cx channel.MCUductance . The subeviously . At −120open iCa . BecausePo) was significantly decreased ~2–3 fold in MICU1-KO versus WT mitoplasts, depending on the transmembrane voltage , in nominally Ca2+-free conditions (MICUs in Ca2+-free state)  when Na+ was the permeant ion. These sub-conductance states were the same in WT and MICU1-KO  . SimilarMICU1-KO . Howeverting ion . Accorditoplasts . This coenotypes .2+-bound state, the MICUs potentiate MCU current by increasing the open probability of the MCU/EMRE pore. In the absence of Ca2+, the MICUs do not appear to affect the pore activity.These results demonstrate that in the Ca2+) is essential for the proper function of several mitochondrial enzymes, its excessive accumulation inhibits oxidative phosphorylation and causes toxicity  in the presence of 5 mM [Mn2+]i. IMn disappeared in MCU-KO and EMRE-KO, confirming that Mn2+ current was solely mediated by MCUcx  than ICa via MCUcx, as was also shown previously  was not affected in MICU1-KO  and MICU1-KO (156 ± 20 μM). Taken together, these findings indicate that Mg2+ exerted its inhibitory effect at the MCUcx pore . However, Mg2+ could bind to the outermost and wider Asp binding site (site 1) of the selectivity filter even with a hydration shell. Such Mg2+ binding would not allow Mg2+ permeation, but would occlude the pore.We first studied how cytosolic Mg [Ca2+]i . The res [Ca2+]i . Thus, w μM or 0 . Thus, tUcx pore  and not hibition  suggestsresidues . Mg2+ is2+ is an MCUcx pore blocker that at the resting [Ca2+]i, would strongly compete with Ca2+ for binding to the selectivity filter, limiting Ca2+ permeation. The Mg2+ occlusion of the MCUcx pore can at least partially explain low mitochondrial Ca2+ uptake at resting [Ca2+]i.In conclusion, Mgcx as cytosolic Ca2+ is elevated and binds to MICU’s EF hands. This potentiation would result in efficient stimulation of the mitochondrial ATP production in response to cytosolic Ca2+ signaling events, when energy demand is increased as shown in neurons  is similar to mammals, MICU1 knockout causes an increase in the Ca2+ uptake ‘threshold’ and a marked decrease in Ca2+ uptake capacity at all [Ca2+]i   , and re-MICU1-KO . In intacx presents formidable difficulties even for the direct electrophysiological analysis. The electrophysiological phenotypes observed may be associated with altered expression levels of other subunits rather than the loss of MICU1. We not only analyzed the expression levels of all MCU complex subunits .2. Cells were maintained by splitting every 48–72 hr at a ratio of 1:5 to 1:10. The MEF cell lines were authenticated by short tandem repeat profiling conducted by Labcorp. The cell lines were free of mycoplasma as determined by PCR based detection (All mouse embryonic fibroblast (MEF) cells with  or withoetection .We used third-generation lentiviral (bi-cistronic) vectors containing the ORF for gene of interest with or without a selection marker . The vectors were generated by VectorBuilder, Inc , and their sequences were confirmed independently by the company and by us. Recombinant cDNA expressing cells were enriched using multiple rounds of FACS or antibiotic selection. In some cases, EGFP was targeted to mitochondria  to identify mitoplasts expressing the recombinant protein of interest during patch clamp experiments.Mice were maintained on a standard rodent chow diet under 12 hr light and dark cycles. All animal experiments were performed with male mice (2–5 month old) according to procedures approved by the UCSF Institutional Animal Care and Use Committee and adhered to NIH standards. C57BL/6J were obtained from the Jackson laboratory. MCU-KO mice were obtained from Dr. Torren Finkel and have been used previously .Gene expression analysis (qRT-PCR) qPCR was performed by Syd Labs . Total RNA was isolated from cells using the RNAeasy Minikit (QIAGEN), and reverse transcribed using the First Strand cDNA Synthesis Kit (Syd Labs). qPCR reactions were performed with the following gene-specific primers (generated by Integrated DNA Technologies):Hprt, Forward Primer 5'-GTCCCAGCGTCGTGATTAGC-3'GTGATGGCCTCCCATCTCCT-3'Reverse Primer 5'-Mcu, Forward Primer 5’-AAGGGCTTAGCGAGTCTTGTC-3'GGGTGCTGGTGTGTTAGTGT −3'Reverse Primer 5’- Mcub, Forward Primer 5’-CCACACCCCAGGTTTTATGTATG-3'ATGGCAGAGTGAGGGTTACCA-3'Reverse Primer 5’-Smdt1, Forward Primer 5’-ATTTTGCCCAAGCCGGTGAA-3'CCTCAAGCAGAGCAGCGAAG-3'Reverse Primer 5’-Micu1, Forward Primer 5'-CTTAACACCCTTTCTGCGTTGG-3'AGCATCAATCTTCGTTTGGTCT-3'Reverse Primer 5'-Micu2, Forward Primer 5'-CTCCGCAAACAGCGGTTCAT-3'TGCCAGCTTCTTGACCAGTG-3'Reverse Primer 5'-Micu3, Forward Primer 5’-GTAAGGTCAGAGCACGCAGAA-3'TTTCCTGTTGGACGCTGACAA −3'Reverse Primer 5’-Hprt was selected as the reference gene.cDNA  samples were pre-amplified for 12 cycles using ABsolute qPCR SYBR Green Low ROX Mix (ThermoFisher). qPCR reactions were performed using an Agilent MX3000 (Fluidigm) with 40 cycles of amplification . Ct values were calculated by the Real-Time PCR Analysis Software (Fluidigm). Relative gene expression was determined by the ΔCt method. Knockout MEF cell lines were generated using the CRISPR/Cas9 method . All knoMcu, TGGCAGCGCTCGCGTCGAGA GGGSmdt1, GAGTGTCCCGACATAGAGAA AGGCTTACACTCCCACTAGGTTA AGGMicu1, TCACTTTTAGATGCTGCCGG TGGCTGCAAGTACCGGTCTCCTG TGGMicu2, CGTTCGGGAGCCCTCGCGCG CGGGGGCGCTTCCGCAAAGATGG CGGMicu3, GGGCGAGCTGAGCATCGCGG CGGCCGGGGCCGCTAGCTCCGAG GGG6 cells and 1 µg of two different gRNA-Cas9 plasmids. Puromycin was used for enrichment of transfected cells, and serial dilution was performed to select single-cell clones. A stable homozygous knockout cell line was confirmed by PCR amplification of the targeted region, cloning into a pUC19 vector, and sequencing showing that either a frameshift or large deletion had occurred in the targeted region of the gene (MEFs were transfected with the Cas9 gRNA vector (Addgene: PX459) via electroporation (Invitrogen Neon transfection system) using the following parameters: 1×10the gene . All knothe gene . The priMcu, Forward Primer TAGAAGCTTTCCACTGCTCTGATTGATCTTGATGTGAATTCGAGCTGCTTTGGAATGAGACReverse Primer Smdt1, Forward Primer GTGAAGCTTGGGATCAGTAGTCCATTGGAGGAGGAGAATTCAGTGAGAGTTCCTGTGGTATGReverse Primer Micu1, Forward Primer TTTAAGCTTGATTCCTTTGAGTTATAAGTAGCAAAGAATTCAGCAAAGAAATTCTGATGTAReverse Primer Micu2, Forward Primer ACCAAGCTTGAACGTCGAGGAAGCAGCCACAGGAGAATTCTCCATCCACCAGGTGGGCAGReverse Primer Micu3, Forward Primer CGCAAGCTTCTCGCGAGATTTCGGCCCGCCAGGAGAATTCTCCATCCACCAGGTGGGCAGReverse Primer Mitoplasts were isolated from MEFs using methodology previously described . BrieflyMitoplasts were produced from mitochondria using a French press. Mitochondria were suspended in a hypertonic solution containing 140 mM sucrose, 440 mM D-mannitol, 5 mM HEPES, and 1 mM EGTA (pH adjusted to 7.2 with Trizma base) and then subjected to a French press at 1200–2000 psi to rupture the outer membrane. Mitoplasts were pelleted at 10,500× g for 15 min and resuspended for storage in 0.5–1 ml of solution containing 750 mM KCl, 100 mM HEPES, and 1 mM EGTA (pH adjusted to 7.2 with Trizma base). Mitoplasts prepared and stored with this method contained the same amount of auxiliary MICU1 and MICU2 subunits as compared to intact mitochondria  plating on 5 mm coverslips pretreated with 0.1% gelatin to reduce mitoplast adhesion.Whole mitoplast currents were measured as described previously . Gigaohmobserved upon application of voltage steps were removed from current traces.All indicated voltages are on the matrix side of the IMM (pipette solution), relative to the cytosolic side .Solution B was used to measure Na+ or Mn2+ currents and contained: 110 Na-gluconate, 40 HEPES, 1 EGTA, 5 EDTA, and 2 mM NaCl (pH 7.0 with Tris base).Solution C was used to measure outward Ca2+ currents (the MCU rectification experiments) and contained: 130 mM tetramethylammonium hydroxide (TMA), 100 mM HEPES and 2 mM CaCl2 (pH 7.0 with D-gluconic acid).2+ currents, the bath solution was formulated to contain only 150 mM HEPES  and different dilutions of CaCl2 from a 1 M stock (Sigma) . The bath solution for measuring inhibition of Na+ current by cytosolic Ca2+ contained: 110 mM Na-gluconate, 40 mM HEPES, and 10 mM EDTA  and varying amounts of CaCl2 were added to the bath solution to achieve the free [Ca2+] calculated using the MaxChelator program .To measure whole-mitoplast Ca (Sigma) . The con2+]i from virtual zero (control solution) to 1 mM was achieved using a commercially available fast solution exchange system . It was interfaced with our pClamp acquisition software in order to precisely time the steps during solution change. The timing (τ ~0.4 ms) for solution exchange was judged by the current changes because of a junction potential difference using solutions with different ionic strengths.A rapid exchange of  (upstroke) was detected using a script written in Python and manually checked afterwards. Briefly, the fluorescence signal was smoothed by applying a second-order zero phase digital Butterworth filter with an optimal cutoff frequency as previously described , or RB supplemented with 40 μM EGTA (for measurements carried out above 3 μM [Ca2+]i). After final centrifugation step at 3200 g, protein concentration was determined by Lowry assay.Briefly, mitochondria were isolated from MEF cells using differential centrifugation as described above. The mitochondrial pellet was resuspended in resuspension buffer (RB) supplemented with 2 mM EGTA and 2 μM of Fura-2-acetoxymethyl ester (Fura-2 AM) and kept at room temperature for 10 min to allow loading of Fura-2 into the mitochondrial matrix. The RB buffer contained: 100 mM KCl, 50 mM MOPS, 1 mM MgCl2+ influx were carried out using a BMG LABTECH CLARIOstar plate reader as described before , supplemented with energetic substrates , and 1 μM TMRM. The uAB and all the stock solutions were made with analytical-grade deionized water  and contained less than 50 nM of residual [Ca2+] (measured daily). After 3 min of incubation with substrates, assays were initiated by injection of 100 μl of Ca2+ stock to bring the final volume to 200 μl. TMRM  and Fura-2  fluorescence were measured, along with Fluo-4 or Fluo4-FF  within the same well for 35 s. To measure MCU Ca2+ flux (JMCU), two protocols were used. Protocol 1 ([Ca2+] range ≤ 3 μM): here, Fluo-4 (3 μM) is the single significant buffer of extra-mitochondrial Ca2+ . Protocol 2 ([Ca2+] range from 4 μM to 25 μM): here mitochondria were suspended in 40 μM EGTA and 1 μM Fluo4-FF was used. Total Ca2+ influx (J) is taken as the first derivative of the linear fit to the measured total extramitochondrial [Ca2+] over the first 20 s of each experiment. The total Ca2+ conductance of the IMM (G) was obtained from the simultaneous measurements of J, [Ca2+]i, [Ca2+]m and ΔΨm according to the typical Hodgkin–Huxley model  where CEa2+ is the Nernst reversal potential for Ca2+ obtained from simultaneously measured [Ca2+]i, and [Ca2+]m. Measured J was converted to I using the Faraday constant  with 200 nM TMRE. Mitochondrial concentration in the assay buffer was 0.25 mg/ml. TMRE fluorescence were measured at 550/570 nm and 570/589 nm  with using a Biotek Synergy H4 plate reader, and the fluorescence ratio between two fluorescence was calculated. After 5 min incubation of mitochondria in the assay medium, assays were initiated by injection of 0.5–1 μl of EDTA , RuR  or FCCP , and the ratio change within 5 min after the injection was evaluated. The ratio change induced by EDTA and/or RuR was normalized with that by FCCP.Membrane potentials in intact mitochondria were evaluated with TMRE using previously described method . MitochoMitochondria or mitoplasts were isolated from MEFs deficient in the MCU subunit but stably expressing Flag-tagged MCU. Mitochondrial fraction from wild type cells (without MCU-FLAG) was used as negative control. Isolated mitoplasts (but not mitochondria) were incubated in 750 mM KCl for 30 min before solubilization. Briefly, 300 µg of protein lysate was solubilized with 500 µl of lysis buffer  for 30 min at 4°C. Lysates were cleared by spinning at 20,000× g for 10 min at 4°C. Cleared lysates were incubated with anti-Flag M2 affinity gel (Sigma A2220) for 2 hr at 4°C. Immunoprecipitates were washed with 1 ml of lysis buffer three times and boiled in 20 μl of Laemmli buffer (without β-mercaptoethanol). One-third of the immunoprecipitate was loaded onto a 4–20% gradient SDS-PAGE gel for detection of the indicated proteins by Western blotting. Flow-through fraction was also collected and analyzed in the same gel.For western blot analysis, MEFs or isolated mitochondria/mitoplasts were lysed in radioimmunoprecipitation assay (RIPA) buffer  and a cocktail of proteases inhibitors). Lysates were resolved by SDS-PAGE; transferred to PVDF membrane (Millipore); and probed with anti-MCU , anti-EMRE , anti-HSP60 , anti-VDAC , anti-MICU1 , anti-MICU2 , anti-MICU3 , and anti-TOM20 . Anti-MICU1 antibody produced a non-specific band near its monomeric molecular weight (~50 kDa), so samples were prepared in Laemmli buffer without β-mercaptoethanol to detect MICU1 homo- or heterodimers (~100 kDa).-value was determined with the methods as indicated in the corresponding Figure legends.Data are presented as mean ± standard error of the mean (SEM), as specified in the Figure legend. Statistical analysis was completed in Excel or Origin 9.6. All experiments were performed in triplicate or more. Statistical significance at an exact p public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Acceptance summary:cx) regulate calcium uptake by mitochondria, a process that serves to match the rate of ATP generation to cellular metabolic needs. Based on direct electrophysiological recordings of MCUcx, the authors find that the MICU1 subunit potentiates channel activity in a calcium-dependent manner but does not block the channel at low calcium levels, challenging current models of MCU regulation. This work will be of significant interest to biophysicists and cell biologists interested in mitochondrial biology, bioenergetics, and ion channel and calcium signaling mechanisms.This paper examines the roles and mechanisms of how subunits of the mitochondrial calcium uniporter complex .Thank you for submitting your article "The Mechanism of MICU-Dependent Gating of the Mitochondrial CaThe reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:2+. This is supported by Na+ flux through MCU under conditions of 0 Ca2+ and Mg2+ to allow Na+ permeation . However, Ca2+ uptake at low levels of Ca2+ is apparently higher in MICU1-/- cells compared to WT cells , consistent with the idea that MICU1 does inhibit MCUcx at resting intracellular Ca2+. A weakness of the manuscript is that no mechanism is investigated or proposed for this effect of MICU1. Rather, this result is explained away by vague arguments that MICU1 KO might change Ca2+ efflux, buffering, pH, or OMM permeability (lines 365-7). A more serious attempt should be made to understand the basis of this effect; new experimental evidence or a more detailed mechanism based on published work should be presented to explain how MICU1 could prevent the rise of free mitochondrial Ca2+ in the presence of low rate of influx. A potential plausible mechanism is that one particular EF hand is responsible for this low Ca2+ blocking effect while another mediates the high Ca2+- dependent potentiation of MCUcx open probability.1. A major conclusion of the paper is that MICU1 does not block the MCU pore at low levels of Ca2+ uptake by mutations predicted to disrupt electrostatic interactions in the plug structure .Also, the authors imply that structural evidence for MICU1 blocking the pore was only obtained for the monomeric complex, and that the more natural dimeric complex did not show block (l. 408-418). This is not strictly true . Also, while it is possible that low ionic strength could lead to artifactual plugging in the structures (l. 421-3), to be fair it should also be noted that the plug model is consistent with increased Ca+ flux through MCU in the absence of Ca2+ and Mg2+, arguing against its function as a pore blocker . However, it is possible that the removal of all divalent cations to permit Na+ permeation prevents MICU1 from plugging the pore . Given the number of previous functional and structural studies that support a blocking role for MICU1, it is important to rule out a possible dependence on Mg2+. If Na+ can permeate in the presence of Mg2+, then the Na+ current measurements or mitochondrial depolarization experiments could be repeated with Mg2+ present. If this is not possible, then Mg2+ could be removed in the Ca2+ uptake experiments  to see whether 0 Mg2+ phenocopies the MICU1 KO effect on uptake at low Ca2+.2. Strong evidence in mitoplasts and intact mitochondria shows that MICU1 does not affect Na3. Interpretation of mut-EF-MICU2 data . Authors indicate dominant negative effect of mut-EF-MICU2 is due to displacement of MICU1 from MICU1 homodimers. Presumably, WT MICU2 also displaces the MICU1 from MICU1 homodimers. The results provided suggest that MICU1 homodimers are functionally equivalent to MICU1/MICU2 heterodimers. It is shown that MICU2 knockout increases MICU1 homodimers. What happens with MICU2 expression in MICU1 knockout? Is the ~100 kDa band in the MICU2 blot  actually representative of MICU1/MICU2 heterodimer?4. The experiments on kinetics of the MICU1 potentiation  need a clearer interpretation.2+ current on addition of Ca2+ are not affected by MICU1. Rather than describing potentiation as "instantaneous" , it may be more meaningful to describe the result in a physiological context; e.g., potentiation is fast enough that MICU1 and mitochondria will faithfully track changes in cytosolic [Ca2+] which are typically quite slow (order of tens-hundreds of msec).a) The kinetics of Cab) The interpretation of Figure 3E depends on the solution switching speed not being rate-limiting relative to the current response. The SF-77B perfusion system is described as piezoelectric (l. 748-753), but to my knowledge this is driven by a stepper motor, and it is not clear how you could get such a fast switching time (tau = 0.4 ms) from this system. Thus, it might be rate-limiting for the response. It may be helpful to show the time course of solution exchange superimposed on the traces in Figure 3E. If the solution exchange is rate-limiting for these responses, then one cannot draw any conclusions about the speed of MICU1 potentiation response.5. The lack of outward flux through MCU supports the conclusion that it is a one-way portal , but alternatives should also be considered. Is it possible that outward flux through MCU requires prolonged depolarization such as would occur in vivo? This could be easily tested in whole-mitoplast recordings using a holding potential that mimics the depolarized voltages used in the previous cited studies.2+ and the MICU does not plug the channel assume that whole-mitoplast recordings preserve the normal regulation of the channel. It is likely that diffusible molecules are lost by dialysis into the recording pipette. The authors should discuss whether and why they think this is not a problem.6. The conclusions that MCU is not regulated by matrix Ca Essential revisions:2+. This is supported by Na+ flux through MCU under conditions of 0 Ca2+ and Mg2+ to allow Na+ permeation . However, Ca2+ uptake at low levels of Ca2+ is apparently higher in MICU1-/- cells compared to WT cells , consistent with the idea that MICU1 does inhibit MCUcx at resting intracellular Ca2+. A weakness of the manuscript is that no mechanism is investigated or proposed for this effect of MICU1. Rather, this result is explained away by vague arguments that MICU1 KO might change Ca2+ efflux, buffering, pH, or OMM permeability (lines 365-7). A more serious attempt should be made to understand the basis of this effect; new experimental evidence or a more detailed mechanism based on published work should be presented to explain how MICU1 could prevent the rise of free mitochondrial Ca2+ in the presence of low rate of influx. A potential plausible mechanism is that one particular EF hand is responsible for this low Ca2+ blocking effect while another mediates the high Ca2+- dependent potentiation of MCUcx open probability.1. A major conclusion of the paper is that MICU1 does not block the MCU pore at low levels of Ca2+ uptake by mutations predicted to disrupt electrostatic interactions in the plug structure .Also, the authors imply that structural evidence for MICU1 blocking the pore was only obtained for the monomeric complex, and that the more natural dimeric complex did not show block (l. 408-418). This is not strictly true . Also, while it is possible that low ionic strength could lead to artifactual plugging in the structures (l. 421-3), to be fair it should also be noted that the plug model is consistent with increased Ca2+], measurements of mitochondrial Ca2+ uptake do not detect a Ca2+ influx, while electrophysiological measurements find no evidence for conduction occlusion of MCUcx by MICUs. Our short answer is that measurements of mitochondrial Ca2+ uptake do not exclusively measure conductance by the MCUcx while the electrophysiological recording presented here do. We examined MCU currents, a direct measurement, and provide new evidence that could partially explain why the net uptake (uptake minus efflux) is low. In newly added experiments  we now show that the conduction pathway of MCUcx is not plugged by MICU1-- even at physiological levels of [Mg2+]. Instead, Mg2+ strongly blocks the selectivity filter of the pore. In addition, the unitary conductance of an open MCUcx channel at 100 nM [Ca2+] is extremely low. The MCUcx flux is thus limited not only because of the low concentration of the conducting ion but also because of Mg2+ block. This allows Ca2+ efflux machinery to effectively compete with MCUcx-mediated Ca2+ uptake at low cytosolic [Ca2+] to reduce net Ca2+ accumulation to nearly zero.The reviewers raise an important question, why there is an apparent discrepancy between the findings of two methodological approaches. Why at low levels of cytosolic [Ca2+ uptake at low levels of [Ca2+]?” This is a question that our study does not directly examine. However, we can speculate -- based on recent other studies  -- that knocking out MICU1 affects multiple mitochondrial systems and not only the MCUcx. Here, we investigated the specific role that MICU1 play as part of the channel complex and do this by directly examining the MCUcx current. We see no evidence that supports the hypothesis that MICU1 acts as a plug of the pore. Instead, our work shows that at elevated levels of [Ca2+], Ca2+ binding to the EF hands of the MICU1 works to double the open probability of MCUcx. The binding of Ca2+ to the EF hands of MICUs, or any consequential effects of this binding occur at levels of [Ca2+] that are higher than 100 nM. Indeed, the available titration data demonstrates that the affinity of the MICUs for Ca2+ is not high enough for EF hands occupancy to occur at 100 nM. Even for MICU1, which has the highest affinity for Ca2+ of all MICUs, no significant binding occurs at 100 nM (resting cytosolic Ca2+), and complete saturation of binding would only occur around 3-6 μM  . Thus, the quantitative evidence also suggests that Ca2+ occupancy of the EF hands at resting cytosolic Ca2+ is too low to explain any profound occlusion.“Why does knocking out MICU1 tends to increase the net mitochondrial Cacx. However, the occluded dimeric complex represented only ~10% of the total number of analyzed particles in the absence of Ca2+. The low prevalence of the dimeric complexes can be purely due to the experimental limitations, but, regardless, a more thorough analysis of occlusion in this native form of MCUcx is needed. Also, such structural analysis should be performed in the presence of physiological concentrations of Mg2+, while so far Mg2+ was absent in all structural studies of the MCUcx holocomplex. We have rewritten the section discussing MCUcx structures to reflect these changes.We agree with the reviewers comment that Wang et al., 2020b showed the possibility of the occlusion in the native dimeric form of the MCUAlthough the occlusion model appears to be consistent with some of the mutations that disrupt electrostatic interactions in the plug structure, all such studies were performed using indirect assessment of MCU function. Such mutations can cause MICUs loss-of-function effects (that is not necessarily loss of occlusion) and could lead to activation of compensatory mechanisms that create an appearance of “the loss of the threshold”, similar to that observed in MICU1 knockout. To interpret the structures correctly, reliable direct functional data is much preferred, as it has always has been the case in the ion channel field.+ flux through MCU in the absence of Ca2+ and Mg2+, arguing against its function as a pore blocker . However, it is possible that the removal of all divalent cations to permit Na+ permeation prevents MICU1 from plugging the pore . Given the number of previous functional and structural studies that support a blocking role for MICU1, it is important to rule out a possible dependence on Mg2+. If Na+ can permeate in the presence of Mg2+, then the Na+ current measurements or mitochondrial depolarization experiments could be repeated with Mg2+ present. If this is not possible, then Mg2+ could be removed in the Ca2+ uptake experiments  to see whether 0 Mg2+ phenocopies the MICU1 KO effect on uptake at low Ca2+.2. Strong evidence in mitoplasts and intact mitochondria shows that MICU1 does not affect Na2+ on Ca2+ currents to answer this question. Please see the new Figure 7. These experiments examine how Mg2+ affects the Ca2+ conduction through MCUcx. There are two main conclusions. First, Mg2+ interacts with the selectivity filter within the pore of MCUcx to occlude Ca2+ permeation. This effect is completely MICU-independent. Second, the Ca2+-dependent potentiating effect of MICUs on ICa does not depend on Mg2+.We have added a set of new electrophysiological experiments to address the effect of Mg2+ uptake, but focus specifically on MCUcx activity. The increase in net mitochondrial Ca2+ uptake in MICU1-KO vs WT was observed both in the presence  or absence of Mg2+ . Thus, the putative occlusion of the MCU pore by MICUs, if it exists, would be a Mg2+-independent phenomenon.We would also like to reemphasize that in this study we did not center our investigation on the net mitochondrial Ca2+ block has, in limiting MCUcx flux at low Ca2+. We also emphasize the importance of reevaluating the structure of MCU holocomplex in the Mg2+ bound conformation. We thank the reviewers for prompting this addition.With these new results included in the manuscript, we revised the results and the Discussion sections. We now highlight the impact that physiological Mg2+ but also Mg2+.Per reviewers’ request, we now clearly indicate in the text that the use of EDTA removes not only Ca3. Interpretation of mut-EF-MICU2 data . Authors indicate dominant negative effect of mut-EF-MICU2 is due to displacement of MICU1 from MICU1 homodimers. Presumably, WT MICU2 also displaces the MICU1 from MICU1 homodimers. The results provided suggest that MICU1 homodimers are functionally equivalent to MICU1/MICU2 heterodimers. It is shown that MICU2 knockout increases MICU1 homodimers. What happens with MICU2 expression in MICU1 knockout? Is the ~100 kDa band in the MICU2 blot  actually representative of MICU1/MICU2 heterodimer?MICU1 is the primary subunit responsible for tethering of other MICUs (MICU2 and MICU3) to the MCU/EMRE pore. In the absence of MICU1, MICU2 cannot bind to the pore. This has been shown in many previous studies both biochemically and structurally. MICU2 expression remains unchanged or a significant amount gets degraded in the absence of MICU1 .The 100 kDa band in the Figure 1—figure supplement 1H  represents MICU2 in the MICU1/MICU2 heterodimer. Although the protein samples in the Figure 1—figure supplement 1H were prepared under the reducing conditions (i.e. with β-mercaptoethanol), we observed bands near the molecular weights corresponding to both the monomeric and heterodimeric forms. For detection of MICU1, the samples were prepared under non-reducing conditions, so the band was always observed at the molecular weight corresponding to the dimeric form. This is mentioned in the Methods section and in the corresponding legends.4. The experiments on kinetics of the MICU1 potentiation  need a clearer interpretation.2+ current on addition of Ca2+ are not affected by MICU1. Rather than describing potentiation as "instantaneous" , it may be more meaningful to describe the result in a physiological context; e.g., potentiation is fast enough that MICU1 and mitochondria will faithfully track changes in cytosolic [Ca2+] which are typically quite slow (order of tens-hundreds of msec).a) The kinetics of Cab) The interpretation of Figure 3E depends on the solution switching speed not being rate-limiting relative to the current response. The SF-77B perfusion system is described as piezoelectric (l. 748-753), but to my knowledge this is driven by a stepper motor, and it is not clear how you could get such a fast switching time (tau = 0.4 ms) from this system. Thus, it might be rate-limiting for the response. It may be helpful to show the time course of solution exchange superimposed on the traces in Figure 3E. If the solution exchange is rate-limiting for these responses, then one cannot draw any conclusions about the speed of MICU1 potentiation response.(a) Thank you for the suggestion. We have modified the manuscript to describe the results in a physiological context as was suggested by the reviewers.(b) Thank you for pointing it out. We acknowledge the error in describing the system. SF77B is a stepper motor. We now show the time-course for the solution exchange in the supplementary Figure 3—figure supplement 2. The time constant of solution exchange was ~0.4 ms. The solution exchange was performed using a thinly-pulled glass theta tubing (double-barreled tube). The mitoplasts  were very close to the perfusion. Because mitoplasts are comparable in size to the tip of the pipette, the rate of solution exchange in actual experiments will be similar.5. The lack of outward flux through MCU supports the conclusion that it is a one-way portal , but alternatives should also be considered. Is it possible that outward flux through MCU requires prolonged depolarization such as would occur in vivo? This could be easily tested in whole-mitoplast recordings using a holding potential that mimics the depolarized voltages used in the previous cited studies.2+) at positive potentials (> 0 mV) show rapid flickering of the outward unitary currents as compared to unoccluded square openings in the inward directions  . However, these recordings demonstrate that efflux of Ca2+ via MCUcx is possible, at least at 105 mM Ca2+ on the matrix side of the IMM. Importantly, these single-channel recording also showed that the Ca2+ efflux via MCUcx does not change with time during prolonged depolarizations. In the whole-mitoplasts experiments presented in the current manuscript, we could not use 105 mM Ca2+ on the matrix side of the IMM (pipette solution), and even 2 mM Ca2+ is barely tolerated, as the membrane integrity is compromised. We cannot exclude a possibility that at 2 mM matrix Ca2+ the outward current via MCUcx is so small that the patch-clamp electrophysiology is unable to resolve it. So, as reviewer implied, we should not claim that Ca2+ efflux via MCUcx is impossible. We now rewrite this part of the manuscript to reflect this.MCU is a highly inwardly rectifying channel. Previous single and multichannel inside-out recordings  suggested that there is a biphasic regulation of MCUcx by matrix Ca2+ ions and this regulation is MICU-dependent. We addressed this possibility but saw no regulation of MCUcx activity by matrix Ca2+ within the physiological range. Regulation of MICU function by matrix Ca2+ appeared plausible at the time when it was still not clear whether MICUs are located on the cytosolic or matrix side of the IMM. Now that the structural data clearly showed MICUs location on the cytosolic face of the IMM, regulation of MICU function by matrix Ca2+ is poorly justified.The manuscript specifically addresses the current model of MICU-dependent occlusion of MCUcx is a large complex and could potentially be regulated by some diffusible matrix molecules. However, these are unlikely to affect MICUs function as MICU proteins are located on the cytosolic face of the IMM.MCU


In the model organism for green algae, Chlamydomonas reinhardtii, Ca2+ signals were reported to have a crucial role in different physiological processes, such as stress responses, photosynthesis, and flagella functions. Recent reports identified the underlying components of the Ca2+ signalling machinery at the level of specific subcellular compartments and reported in vivo imaging of cytosolic Ca2+ concentration in response to environmental stimuli. The characterization of these Ca2+-related mechanisms and proteins in C. reinhardtii is providing knowledge on how microalgae can perceive and respond to environmental stimuli, but also on how this Ca2+ signalling machinery has evolved. Here, we review current knowledge on the cellular mechanisms underlying the generation, shaping, and decoding of Ca2+ signals in C. reinhardtii, providing an overview of the known and possible molecular players involved in the Ca2+ signalling of its different subcellular compartments. The advanced toolkits recently developed to measure time-resolved Ca2+ signalling in living C. reinhardtii cells are also discussed, suggesting how they can improve the study of the role of Ca2+ signals in the cellular response of microalgae to environmental stimuli.Calcium (Ca 2+ signalling machinery of Chlamydomonas reinhardtii intracellular compartments, highlighting their contribution to Ca2+ signals and the most advanced tools to study Ca2+ signalling in microalgae.Here we review the Ca A prolonged increase of calcium concentration in the cytoplasm ([Ca2+]cyt) above ~10–4 M causes aggregation of proteins and nucleic acids, and negatively influences the integrity of lipid membranes. Moreover, high [Ca2+]cyt leads to precipitation of phosphates, reducing the availability of this key precursor for nucleotide triphosphates, such as ATP, chosen early on by cells as the energy currency of life , amplitude, and duration are determined by the acting calcium signalling toolkit  of plant cells being important to ensure the transient nature of Ca2+ signals and fast response to environmental stimuli , calmodulins (CaMs), calmodulin-like proteins (CMLs), and calcineurin B-like proteins (CBLs), which act either as primary effectors or as signal relays. These molecules are part of the Ca2+ signalling toolkits in both prokaryotes and eukaryotes , in stress responses to environmental abiotic stimuli, and also in the regulation of photosynthesis  channels, found to play a role in osmotic stress signalling in land plants , which are instead osmoregulatory organelles. The vacuoles of yeasts and higher plants are thought to have evolved from lytic vacuoles of ancestral green algae, whereas CVs appear to have evolved when protists entered freshwater habitats , aquaporins, and vesicular transport  pumps, that could generate a H+ gradient and energize Ca2+/H+ exchange through CAX transporters, sequestering Ca2+ from the cytoplasm  and metal deficiency conditions, accumulating manganese (Mn), zinc (Zn), and copper (Cu)  rich in polyphosphate, in which the estimated total concentration of Ca2+ is ~2–4 M  indicate that a large fraction of the Ca2+ can be quickly mobilized from C. moewusii EDBs, suggesting the presence of a delicate and balanced equilibrium between soluble and insoluble forms of calcium and phosphate , producing inositol-3-phosphate (InsP3). Vacuoles in the yeast Saccharomyces cerevisiae, similar in size, content, and electron density to EDBs, have been shown to release Ca2+ in an InsP3-dependent manner  homologue  is one of the most predominant forms. Chlamydomonas reinhardtii cells express a CR homologue (CRT2) that putatively localizes to the ER  protein family contribute to cytosolic Ca2+ elevations, especially Msy2, by Ca2+ release from the cortical ER after a hypo-osmotic shock  also allows changes in luminal ER [Ca2+] to regulate the opening of a plasma membrane Ca2+ channel, modulating cytosolic [Ca2+] and influencing several biological processes. This system is composed of an ER Ca2+ sensor, STIM, and a plasma membrane Ca2+ channel regulated by STIM, called Orai. In the genome of C. reinhardtii, one Orai sequence has been identified, showing the preservation of many important sequence features , mainly localized in the ER and Golgi apparatus, and P-IIB autoinhibited Ca2+-ATPases (ACAs), found mainly in the plasma membrane but also in the ER, vacuoles, and chloroplast membranes  is bound to the negatively charged thylakoid membranes or to Ca2+-binding proteins, keeping the resting free [Ca2+]stroma as low as 150 nM to avoid the precipitation of phosphates  and BICAT 2  in Mn2+ homeostasis rather than in Ca2+ transport (C. reinhardtii chloroplasts (CGLD1)  , where i2+ signals and Ca2+-permeable channels/transporters have been extensively performed in higher plants whereas, in C. reinhardtii, the presence and identification of stimulus-specific chloroplast Ca2+ signals and transients, together with the Ca2+ channels and transporters responsible, remains unexplored. The most recent related studies in C. reinhardtii were addressed to study Ca2+-dependent regulation of photosynthesis and its associated photoprotective mechanisms, taking advantage of the presence of one single large chloroplast rather than many (reviewed by 2+-sensing receptor (CAS) of C. reinhardtii  which is an important actor for high light acclimation and photoprotection in this organism  knockout mutant, unable to grow at ambient CO2, has also proposed CAS as a Ca2+-mediated regulator of CO2-concentrating mechanism (CCM)-related genes  in the chloroplast CCM  in C. reinhardtii, in both the PGRL1/PGR5-related and the NAD(P)H dehydrogenase (NDH)-dependent pathway (CrCAS directly interacts with Proton Gradient Regulation Like 1 (PGRL1) and Anaerobic Response 1 (ANR1), in a multiprotein complex, thus being required with Ca2+ in the modulation of qE and CEF , characterized by two EF-hands motifs and suggested to have a Ca2+ binding capability, that might modulate its activity , has been identified and characterized in C. reinhardtii  upon Ca2+ binding, was suggested as a crucial point of interconnection of redox and Ca2+ signalling in the chloroplast, probably translating alteration of Ca2+ and the redox state into cellular signalling , it could in fact respond to increases of stromal [Ca2+], accelerating the rate of PRX1 reduction and ultimately modulating stromal redox poise. Moreover, in vivo analysis revealed that CEF is induced on reducing or removing CRX activity, which could be caused by enhanced ROS production in light, thus suggesting a further indirect role of Ca2+ in the regulation of algal photosynthesis  has been identified but not characterized yet, namely GLR1  signalling under abiotic stress, thus opening up a potential plastidial localization also of C. reinhardtii GLR1  have been shown to be intimately linked to cytosolic Ca2+ transients during hyperosmotic stress. In C. reinhardtii, three putative KEA transporters have been identified, each one of a different subtype, whose function and localization still remain to be defined  1 and 2, light-gated H+- and Ca2+-permeable channels able to initiate mainly Ca2+ fast inward-directed complex currents in the eyespot region  presumably through voltage-dependent anion channels , found to allow fluxes of metabolites and Ca2+ in both animals and plants  and ASC2 (Cre05.g241950) (2+ transport across the OMM toward the intermembrane space (Cag241950) , that co2+ ions, which can passively flux across the IMM into the matrix through specific channels and/or transporters. Once in the mitochondrial matrix, Ca2+ can be bound by proteins and/or inorganic acids, allowing the accumulation of large amounts of total sequestered Ca2+, maintaining the resting [Ca2+] in the matrix at ~100–200 nM  is present in C. reinhardtii , shows 43.1% sequence identity with the Ca2+-binding CaM-like 38 (CML38) protein of Arabidopsis, whereas MOT6 (Cre03.g197500) interestingly shows 36% sequence identity to a human calcyphosin-like protein, a Ca2+-binding protein that may play a role in the regulation of Ca2+ transport  and MOT6 in Ca2+ signalling could be represented by the analysis of cytosolic and mitochondrial Ca2+ dynamics in C. reinhardtii mutant strains lacking one of these putative transporters or sensors. A further possible strategy to elucidate the role of mitochondria in algal Ca2+ signalling processes might be a forward genetic screening on C. reinhardtii mutants impaired in mitochondrial Ca2+ homeostasis in order to identify the molecular players involved in this process.In mammals, mitochondrial electron transport chain (ETC) reactions, coupled with proton movement across the inner mitochondrial membrane (IMM), generate a well-defined proton motive force (pmf), composed of a proton gradient and an electric component, reaching values around –180/–220 mV . Such annhardtii , whereasd AtcMCU . New evi2+ cycle . Furtherand MOT6 , 2 , and nicotinic acid adenine dinucleotide phosphate (NAADP), like Ins3PR and ryanodine receptors (Ryrs)  from Medicago truncatula, and the homologues CASTOR and POLLUX from Lotus japonicus, have been identified as cation channels involved in the nuclear Ca2+ spiking typical of the early stage of the symbiotic signal transduction pathway, resulting in downstream symbiosis-related gene expression  can be distinguished as efficient tools for in vivo measurements of both Ca2+ concentrations and fluxes. A study of Ca2+ dynamics in living cells was initially performed through fluorescent Ca2+-sensitive dyes , which have been successfully loaded into animal, plant, and even algal cells  to study the role of cytosolic [Ca2+] elevation in the deflagellation process of C. reinhardtii cells (C. reinhardtii exploited the versatility of Ca2+-responsive Oregon Green (OG)-BAPTA dextran to evidence the role of Ca2+ signalling in the gliding motility process and osmotic stress responses  microscopy -related proteins (i.e. Cameleons) and display high spatio-temporal resolution and sensitivity, thus being extremely useful in Ca2+ signalling studies. Cameleons [e.g. yellow Cameleon 3.6 (YC3.6)] are Förster resonance energy transfer (FRET)-based indicator proteins, which harbour cyan and yellow fluorescent proteins  linked together by the Ca2+-binding protein CaM and the CaM-binding peptide M13 , were also localized to different compartments simultaneously, also exhibiting in some cases significantly higher signal change compared with YC3.6 in response to several stimuli , accompanied by other Ca2+-related molecular players conserved among Viridiplantae and other ‘animal-like’ Ca2+ signalling components such as Ca2+ transporters and Ca2+-binding proteins , a genome-wide indexed library of mapped insertion mutants, could also represent a powerful resource to accelerate this process (2+-related genes into pathways and the discovery of new uncharacterized ones. Mechanisms of intracellular Ca2+ signal transduction were also not completely elucidated, including how influx and efflux through these molecular players are regulated in a concerted manner to translate specific information into unique Ca2+ transients. To some extent, many Ca2+-based signal transduction networks have been reported in land plant and animal cells, including the possible role of each subcellular compartment in the shaping of the global Ca2+ signal. Conversely, our understanding of intracellular and organellar Ca2+ signalling in algae, and in particular in the green model alga C. reinhardtii, remains in its infancy. A useful approach to understand how different organelles could act as a source of Ca2+, for example, could be represented by the study of C. reinhardtii mutant strains lacking specific Ca2+ transporters/channels, together with pharmacological approaches using Ca2+ chelators or inhibitors of differentially distributed Ca2+ channels (C. reinhardtii mutant strains stably expressing selectively targeted GECIs, will allow the imaging of cytosolic and intraorganellar [Ca2+] transients, possibly leading to the characterization of the depleted channels/transporters and to the definition of their role in the generation of different cytosolic and organellar stimulus-induced Ca2+ transients. A clear knowledge of the molecular players involved in C. reinhardtii organellar Ca2+ signalling, together with a precise understanding of their role in the regulation of cytosolic and intraorganellar Ca2+ fluxes, could really help to clarify the role of each organelle in the shaping of intracellular Ca2+ dynamics and consequently in the regulation of each Ca2+-based biological process. Together these studies could elucidate many aspects of the functioning of Ca2+-dependent signalling pathways in C. reinhardtii, providing new important insights both into their evolution among eukaryotes and into the understanding of their role in the perception and response to environmental stimuli.Several pieces of evidence support a crucial role for CaChlorella vulgaris, Dunaliella salina, and Haematococcus lacustris, toward the optimization of biomolecules and biomass production in a microalgal biorefinery concept.Microalgae currently represent a renewable and sustainable source of biomass and highly valuable metabolites, whose composition and production are highly affected by environmental factors, including biotic and abiotic stresses (


Thus, we used single-vesicle imaging to quantify the extent to which Ca2+ regulates nanoscale vesicle clustering mediated by α-Syn. Our results revealed not only that vesicle clustering required α-Syn to bind to anionic lipid vesicles, but also that different concentrations of Ca2+ exerted different effects on how α-Syn induced vesicle clustering. In particular, low concentrations of Ca2+ inhibited vesicle clustering by blocking the electrostatic interaction between the lipid membrane and the N terminus of α-Syn, whereas high concentrations promoted vesicle clustering, possibly due to the electrostatic interaction between Ca2+ and the negatively charged lipids that is independent of α-Syn. Taken together, our results provide critical insights into α-Syn’s physiological function, and how Ca2+ regulates vesicle clustering mediated by α-Syn.Although numerous studies have shown that the protein α-synuclein (α-Syn) plays a central role in Parkinson’s disease, dementia with Lewy bodies, and other neurodegenerative diseases, the protein’s physiological function remains poorly understood. Furthermore, despite recent reports suggesting that, under the influence of Ca Calcium plays a critical role in the clustering of synaptic vesicles (which in a neuron store various neurotransmitters released at the synapse) induced by the protein alpha-synuclein, and different calcium concentrations exert different effects on such clustering. Alpha-synuclein is a key element in the development of Parkinson’s disease and other neurodegenerative conditions: it has been shown to interact with synaptic vesicles under the influence of calcium, but its physiological functions are poorly understood. Jiajie Diao of the University of Cincinnati College of Medicine, USA and colleagues used single-vesicle imaging to examine how calcium regulates vesicle clustering mediated by alpha-synuclein. They found that high calcium concentrations promoted vesicle clustering independent of alpha-synuclein, but low calcium concentrations inhibited vesicle clustering in an alpha-synuclein–dependent manner. The authors believe their findings provide insights into alpha-synuclein’s physiological function. Found to aggregate in a nucleation-dependent manner, α-Syn forms cytotoxic amyloid oligomers and fibrils8. Recent studies have indicated that α-Syn not only binds to the highly curved membranes of synaptic vesicles, but also senses and regulates the curvature of those membranes, which immediately suggests that α-Syn is involved in synaptic vesicle trafficking and its exo- and endocytosis11. However, the physiological function of monomeric α-Syn remains unclear12.α-Synuclein (α-Syn), a presynaptic protein abundantly expressed throughout the central nervous system, is the hallmark of Parkinson’s disease, dementia with Lewy bodies, and other neurodegenerative diseases14. It has also been proposed that α-Syn acts as a bridge between membranes—for example, that two helices each bind to two different synaptic vesicles—the result of which is vesicle clustering15. At the same time, recent studies have suggested that calcium ions (Ca2+) can mediate the interaction between α-Syn and lipid membranes. For instance, Zhang et al. demonstrated that Ca2+ competitively binds to anionic lipids, which triggers the dissociation of α-Syn from membranes17. However, upon finding that Ca2+ increases α-Syn’s binding to anionic lipids via α-Syn’s C terminus, Lautenschläger et al18. proposed that the neutralization of negative charges on residues at the C terminus via the dynamic binding of positively charged Ca2+ facilitates α-Syn’s interaction with phospholipid membranes. They also showed that α-Syn and Ca2+ balance the electrostatic interaction of synaptic vesicles, which facilitates vesicle clustering18. Another recent study revealed that α-Syn amyloid oligomers, acting as multivalent nanoparticles, can cause hemifusion in negatively charged vesicles19. However, how Ca2+ regulates the interaction of α-Syn and lipids remains uncertain.In aqueous solution, α-Syn is an intrinsically disordered protein that binds to small synaptic vesicles via a conserved lipid-binding domain. When binding to a lipid membrane, the N terminus of α-Syn forms either an extended helix structure or two broken structures20. As one option, total internal reflection fluorescence microscopy (TIRFM) is an optical technique for inducing an evanescent wave or field in a limited region of a specimen immediately adjacent to the interface between two media with different refractive indices22. A typical setup for TIRFM is shown in Fig. 22. Thus, TIRFM has benefited studies requiring images of individual particles in specimens with large numbers of fluorophores outside the optical plane of interest22, including during vesicle docking induced by proteins26.Currently, researchers are in great need of a technique for studying the interactions between α-Syn and lipid membranes at the single-particle level2+ regulates vesicle clustering mediated by α-Syn and to investigate the possible underlying mechanism. Among our results, α-Syn induced clustering of anionic lipid vesicles when no Ca2+ interaction occurred. Moreover, vesicle clustering induced by α-Syn was inhibited in the presence of low concentrations of Ca2+, where competitive binding of Ca2+ to lipids altered the electrostatic interaction and, in turn, caused the N terminus of α-Syn to dissociate from the membranes. By contrast, although Ca2+ inhibited the interaction of α-Syn and the lipid membranes, a high concentration of Ca2+ supplied sufficient multivalent ions to bridge vesicles into close contact and enhance vesicle clustering. Our results provide critical insights into α-Syn’s physiological function and the mechanism by which calcium ions regulate vesicle clustering mediated by α-Syn.In our study, we used single-vesicle imaging to quantify the extent to which Ca2+ regulates vesicle clustering with α-Syn, we used transmission electron microscopy (TEM) to image lipid vesicles with α-Syn in the absence of Ca2+. Figure l-serine (DOPS). As shown in Fig. To illustrate how Ca2+ exerted a significant effect on vesicle clustering. At concentrations of 0.5 mM and 2 mM Ca2+, there were fewer vesicle clusters than in the control without Ca2+, which indicates that Ca2+ at concentrations of 0.5 mM and 2 mM inhibits the interaction of α-Syn with vesicles, as depicted in Fig. 2+ outnumbered those in the control, which suggests that higher concentrations of Ca2+ promote vesicle–vesicle interaction mediated by α-Syn. Due to surface variation, the baseline level of vesicle clustering differed significantly across different imaging surfaces; therefore, only experiments on the same imaging slide are directly comparable with absolute counts. To compare the results from different slides, we calculated the normalized value of the clustering counts  of Ca2+ inhibited clustering, whereas a higher concentration  promoted it.To quantify vesicle clustering induced by α-Syn, we performed a single-vesicle assay with two types of fluorescent dye to label different vesicles—free vesicles containing the fluorescent molecule DiI (DiIC18(3)) and immobilized vesicles containing the fluorescent molecule DiD (DiIC18(5)). The detection of vesicle clustering relied upon TIRFM Fig. , which cnts Fig.  based onnts Fig. . A norma2+. We incubated α-Syn with vesicles with 12% PS that were approximately 50 nm in diameter, and that had been labeled with the fluorescent molecule DiD. Although no significant aggregates appeared in the solution with vesicles only  of Ca2+ inhibited clustering, whereas a higher concentration  promoted clustering.To cross-check our results, we performed a bulk vesicle-clustering experiment in the presence of α-Syn and different concentrations of Canly Fig. , vesicleced Fig. , which da2+ Fig. . Such re2+ regulates vesicle clustering, we performed vesicle-clustering experiments in the presence and absence of α-Syn with different concentrations of Ca2+. Moreover, to investigate whether electrically charged lipids affect the clustering interaction in the presence or absence of α-Syn, we prepared lipid vesicles with no electric charge  as a control group. Figure 2+  with and without α-Syn. When α-Syn was absent, no significant difference in vesicle-clustering counts was observed between the vesicles without PS and those with 12% PS, which indicates that low concentrations of Ca2+ do not affect vesicle clustering in the absence of α-Syn. However, when α-Syn was present, clustering counts in vesicles with 12% PS were significantly less than those in vesicles without PS. Thus, the result shown in Fig. 2+ at a low concentration .To gain insights into the mechanism by which Ca2+  without α-Syn, 12% PS increased the vesicle-clustering counts, regardless of whether α-Syn participated in the clustering process  in vesicle clustering may occur via electrostatic interactions between Ca2+ and negatively charged lipids, as shown by earlier findings that multivalent ions may prompt ion bridges and tight coupling between lipid membranes27. Thus, whereas a high concentration of Ca2+ causes many Ca2+ to bind to lipids, and bound multivalent ions can bridge vesicles to form clusters, a low concentration of Ca2+ cannot supply sufficient Ca2+ to drive vesicles into close contact.By contrast, at a high concentration of Caess Fig. . The res2+ and the promotional effect of high concentrations, we recorded the circular dichroism (CD) spectra of α-Syn in CD buffer , as shown in Fig. 2+, which revealed that the content of the α-helix structure of lipid-bound α-Syn decreased. This result confirmed that the inhibitory effect of 0.5 mM Ca2+ on negatively charged vesicle clustering stemmed from the decreased amount of lipid-bound α-Syn. When 10 mM Ca2+ was applied to the lipid-bound α-Syn, the characteristic inverse peaks at 210–222 nm disappeared, and characteristic minima of random coil structures were observed at approximately 200 nm, which implies the nearly complete removal of lipid-bound α-Syn from the negatively charged lipid vesicles. The reason the restored peaks corresponding to a random coil shifted slightly compared with that of free α-Syn is probably that a large amount of Ca2+ bound to the removed α-Syn. Our CD spectrum experiments thus suggest that Ca2+, regardless of its concentration, inhibits the interaction between α-Syn and negatively charged lipid membranes.Having observed the inhibitory effect of low concentrations of Ca2+ affected α-Syn’s interaction with lipid membranes, and to confirm the interaction between α-Syn and lipid vesicles, we performed molecular dynamics (MD) simulations to study the interaction of α-Syn and lipid membranes mediated by Ca2+. The MD simulation method is a powerful approach to study MD and interactions. Previous studies have used MD simulations to study the conformations of α-Syn in membranes28, the dynamics of N-terminally acetylated α-Syn with lipids29, the influence of membrane curvature on α-Syn conformation30, and the effect of Ca2+ on different types of protein structures31. In particular, we tested the binding of α-Syn’s N-terminal domain (amino acids 1–60) to the anionic membrane in different concentrations of Ca2+. As shown in Fig. 2+, the N terminus of α-Syn quickly bound to the membrane with negatively charged lipids, consistent with previous findings that α-Syn prefers to bind to anionic lipids via electrostatic interactions33. Snapshots of the details of α-Syn’s N-terminal binding to the membrane in the absence of Ca2+ can be found in Fig. 2+ at concentrations of 2 mM and 10 mM was in the solution, the N terminus of α-Syn had no contact with the membrane, as shown in Fig. 2+, α-Syn’s N terminus is in close contact with the membrane. However, with Ca2+ at concentrations of 2 and 10 mM, α-Syn’s N terminus clearly distances itself from the membrane’s surface. Figure 2+, whereas none of them formed in the presence of Ca2+. Figure 2+ that competitively bound to negatively charged lipids inhibits the interaction between α-Syn and the membranes. Consistent with the CD experiments  propyl) trimethoxysilane) solution  for 10 min, sonicated for 1 min, and incubated for 10 min again. Each slide or coverslip was then blown dry from the edge using nitrogen gas after washing with Milli-Q H2O followed by methanol. Finally, 100 μl of reaction solution  was placed onto the imaging surface of the slide. Each slide was covered by one clean coverslip to be a set, which was incubated overnight and then rinsed with Milli-Q H2O, dried, and stored at −20 °C for further use.To study vesicle clustering, samples were loaded into a chamber assembled from quartz slides and glass coverslips. It is essential to have clean surfaces for single-vesicle fluorescence imaging. First, the slides and coverslips were rinsed and sonicated using Milli-Q Hl-serine)), 6.4 μl of Bio-PE ) (only for DiI vesicles) and fluorescent dye (100 μl of DiI or DiD) were included in the composition required. The lipid composition of the no-PS vesicles was similar to that of 12% PS vesicles, except for the absence of DOPS. To form a thin lipid film, the mixture in the tube was subjected to vacuum in a ball container and rotated every 30 min until the organic solvent was completely evaporated. Subsequently, 500 μl of HEPES buffer  was added to hydrate the lipid film. Finally, the solution was frozen and thawed at least 5–6 times to obtain vesicles that were filtered through a membrane with 50-nm pores. The diameter distribution of 40–50-nm liposomes was previously measured by TEM34. The dispersed vesicles were stored at −80 °C for further experiments.We used two types of lipid vesicles with fluorescent dyes, i.e., DiI (DiIC18(3)) and DiD (DiIC18(5)), for single-vesicle clustering experiments. Before mixing the lipid reagents, the glass syringe was washed three times with chloroform. The lipid components were added into the glass tube wrapped with a piece of foil with a small hole at the top. For preparation of 12% PS vesicles, 103.8 μl of DOPC , 29.8 μl of DOPE , 48.6 μl of DOPS .DiI and DiD vesicles were detected and imaged after the dyes were excited by a green laser (532 nm) and a red laser (633 nm), respectively. The CCD camera and smCamera program were used to acquire and analyze the images, respectively. The number of vesicles clustered was determined by counting the DiD vesicles whose fluorescent spots were shown in the acceptor channel on each image (45 × 90 μm). Approximately, 15 random locations were imaged and analyzed for each sample channel. The compared results are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) with Tukey’s test was used to determine the statistical significance among different groups. When 2+ solution was added to the mixture of α-Syn and lipid vesicles to observe the effect of Ca2+. The mixture was incubated for more than 20 min before imaging the samples excited by red laser light with confocal microscopy. Imaging was performed using 35-mm glass-bottom dishes (MatTek).To investigate bulk vesicle clustering, the DiD vesicles were dispersed and diluted in 3 wt% mPEG (Mw: 5000) solution to 2 μM. The 1:500 protein–lipid ratio was used to study the bulk clustering in the presence of 4 nM α-Syn. A 10 mM or 0.5 mM Ca2)–thaw (37 °C water bath) cycles. Before use, the vesicles were sonicated three times in low-power mode. Each sonication was performed for 5 min. α-Syn was dissolved in Tris-HCl buffer  to form a protein solution of 2.5 mg/ml, which was dialyzed in 300 ml of CD buffer overnight before use. The final protein solution for detection was 10 μM. The protein-to-lipid ratio in the mixture of α-Syn and lipids used for each test was 1:100. CaCl2 (0.5 mM or 10 mM) dissolved in Tris-HCl buffer was mixed with the corresponding lipid-bound protein solution. The secondary structure of lipid-free or lipid-bound α-Syn was determined by CD spectroscopy (Aviv 215 CD spectrophotometer). Wavelength scans were taken in 0.5-nm steps with a 3-s averaging time and 0.333-s settling time. Spectra were collected from 300 nm to 190 nm at 25 °C in a 0.1-mm path-length quartz cuvette containing samples. The baseline was corrected by the blank (buffer only). Each spectrum was averaged from three repeats. The corrected signal data of each CD spectrum were smoothed with the adjacent-averaging method and then plotted using Origin8.5.The lipid composition of the 12% PS vesicles used for the CD experiments includes 43 mol% DOPC, 45 mol% DOPE, and 12 mol% DOPS. Dried lipid films were rehydrated in deionized water to a concentration of 5 mM. Vesicles were formed after at least six freeze , we applied pzz = 1 bar, pxx = pyy = −28 bar for 15 ns to achieve a packing density of ~1.28 lipids per square nanometer of the membrane, as shown in Fig. 3 TIP3P42 water box, with ~65,000 water molecules and an appropriate number of sodium ions added to neutralize the system.The lipid bilayers were generated by the Membrane Builder online service CHARMM-GUI2+ in solution will significantly bind to the membrane with negatively charged lipids43. To prepare the membrane model with 2 mM and 10 mM Ca2+, we gradually added CaCl2 molecules to the solution, and Ca2+ bound to the membrane, as shown in Fig. 2+ in solution was static at 2 mM and 10 mM for 20 ns, the structures were extracted for simulations of α-Syn binding, as shown in Fig. 13. To reduce the computational demand, only the N terminus of α-Syn  was used in the simulations.According to a previous study, Ca44 with the CHARMM36 force field45. Periodic boundary conditions were applied, and the temperature was coupled to 310.25 K with the V-rescale algorithm46. To maintain the packing density of lipids, the pressure was coupled by the Parrinello–Rahman method as pzz = 1 bar, pxx = pyy = −28 to apply membrane tension47. The LINCS algorithm was applied to constrain the covalent bonds with H atoms48. The time step of the simulations was 2.0 fs. The particle mesh Ewald method was used to calculate the long-range electrostatic interactions49. The cutoff of the nonbonded interactions was set to 12 Å. All graphics and visualization analyses were processed using the VMD package 50.All of the simulations were performed using the GROMACS packageSupporting Materials


Recent experimental evidence indicates that mitochondrial depolarization promotes arrhythmogenic delayed afterdepolarizations (DADs) in cardiac myocytes. However, the nonlinear interactions among the Ca2+ signaling pathways, ROS, and oxidized Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways make it difficult to reveal the mechanisms. Here, we use a recently developed spatiotemporal ventricular myocyte computer model, which consists of a 3-dimensional network of Ca2+ release units (CRUs) intertwined with mitochondria and integrates mitochondrial Ca2+ signaling and other complex signaling pathways, to study the mitochondrial regulation of DADs. With a systematic investigation of the synergistic or competing factors that affect the occurrence of Ca2+ waves and DADs during mitochondrial depolarization, we find that the direct redox effect of ROS on ryanodine receptors (RyRs) plays a critical role in promoting Ca2+ waves and DADs under the acute effect of mitochondrial depolarization. Furthermore, the upregulation of mitochondrial Ca2+ uniporter can promote DADs through Ca2+-dependent opening of mitochondrial permeability transition pores (mPTPs). Also, due to much slower dynamics than Ca2+ cycling and ROS, oxidized CaMKII activation and the cytosolic ATP do not appear to significantly impact the genesis of DADs during the acute phase of mitochondrial depolarization. However, under chronic conditions, ATP depletion suppresses and enhanced CaMKII activation promotes Ca2+ waves and DADs.Mitochondria fulfill the cell’s energy demand and affect the intracellular calcium (Ca Ca2+ waves are known to be promoted by Ca2+ overload under normal (2+ enters into the cytosol from the extracellular space mainly via L-type Ca2+ channels (LCCs) during membrane depolarization, which causes Ca2+ release from the SR, a process called Ca2+-induced Ca2+ release  and taken back to the SR through sarcoplasmic reticulum Ca2+-ATPase (SERCA). Meanwhile, mitochondria, as another Ca2+ store, are involved in intracellular Ca2+ cycling via mitochondrial Ca2+ uniporter  level, enhanced Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation via oxidative stress, Ca2+ influx into the cytosol from the mitochondria, and a decrease in the cytosolic ATP level, etc.Delayed afterdepolarizations (DADs) are abnormal depolarizations during the diastolic phase following an action potential (AP) and could trigger cardiac arrhythmias . DADs ar+) waves , occurrir normal  and pathr normal , long QTr normal , ischemir normal , and catr normal . During se CICR; . Ca2+ ister MCU; , mitochors mNCX; , and thers mNCX; . Besidesnditions , thus im is rare . Howevervia its redox effect  and mitochondria, which contains physiological details of mitochondrial membrane potential, mitochondrial Ca2+ cycling, mPTP stochastic opening and closing, intracellular ROS, and oxidized CaMKII signaling. Using this model, we have investigated the underlying mechanisms of Ca2+ alternans and early afterdepolarizations caused by mitochondrial depolarization and dissected each of the components  CRUs and 5376 (64 14 6) mitochondria. The membrane potential (V) of the cell is described byCm=1 mF/cm2 is the cell membrane capacitance, and Isti is the stimulus pulse with the current density being −80 mA/cm2 and the duration being 0.5 ms.where k12) of RyRs was modeled as follows:The oxidized CaMKII activation and the redox effect of ROS both increase the open probability of RyRs . To incokCaMKII and △kROS are the CaMKII-dependent (Eq. 3) and ROS-dependent components (Eq. 4), respectively. kbase and uk are the rate constants. [Ca2 + ]p is the Ca2+ concentration in the dyadic space of a CRU. [CaMKII]act and [ROS]cyt are the CaMKII activation level and the cytosolic ROS concentration in each CRU, respectively. The increase in CaMKII activation and ROS level increase k12, which in turn increases the open probability of RyRs.where △The formulation of SERCA isfup,ATP, and fup,ROS are ATP and ROS-dependent functions  is a CaMKII dependent function. [CaMKII]act is CaMKII activation level in the cytosolic space of a CRU.0 and C1, and an open state O) Markov model to simulate the stochastic opening and closing of the mPTP. The transition rate from the C0 state to the C1 state, kc0c1, is set as:We used a 3-state , following 3 s without pacing in order to observe Ca2+ waves and DADs. As shown in mPTP, suggesting that mitochondrial depolarization due to openings of mPTP promotes spontaneous Ca2+ release and DADs. Also, when αmPTP is greater than ∼50, the proarrhythmic effect appears to saturate. Under the control condition (αmPTP=1), there is no occurrence of DADs (2+ amplitude is ∼0.8 μM) (mPTP=60), we observed DADs. The corresponding line-scan image clearly shows enhanced spontaneous Ca2+ release as compared to a few scattered Ca2+ sparks under the control condition .We investigated the impact of mitochondrial depolarizations on the occurrence of Caf 300 ms . αmPTP i of DADs . The cyt∼0.8 μM) . Howeverondition . In thismPTP increases the open probability of mPTP, which, in turn, elevates the level of cytosolic ROS. To further identify the role of cytosolic ROS in the genesis of DADs, we performed simulations for a clamped ROS level at the PCL 300 ms. In the free-running ROS case as shown in mPTP values for the clamped ROS . Our results demonstrate that the cytosolic ATP level has a significant impact on the genesis of DADs. However, the depletion of the cytosolic ATP concentration during mitochondrial depolarization is a relatively slow process. Therefore, ATP depletion should not play a central role in the genesis of DADs during the acute phase of mitochondrial depolarization. Thus, similar to CaMKII activation, ATP depletion may only suppress DADs in a much longer time scale.Since the SERCA pump requires ATP for its normal function, a lower level of ATP directly impairs the SERCA pump activity (Eq. 5). However, ATP depletion is a slow process, which is evident from via mPTP opening in the genesis of DADs. The systematic investigation of individual roles, including the cytosolic ROS, mitochondrial Ca2+, CaMKII activation, and the cytosolic ATP, reveals that the redox effect of ROS on RyRs may play an essential role in the occurrence of DADs during the acute phase of mitochondrial depolarization. Furthermore, increasing the MCU activity could promote DADs by increasing the mPTP open probability through mitochondrial Ca2+ dependent kinetics of mPTP. In addition, oxidized CaMKII activation promotes, and ATP depletion suppresses DADs chronically in the condition of mitochondrial depolarization.We used a physiological detailed ventricular myocyte model consisting of a 3D network of coupled CRUs and mitochondria to investigate the roles of mitochondrial depolarization 2+ waves (2+ waves (2+ leaks (Ca2+ spark) amplitude but drastically increases its frequency (2+ handling proteins, such as RyRs and SERCA (2+ waves in experiments during mitochondrial depolarization are the consequences of the combined effects of the above factors. However, what is the primary player remains unclear. Here, by using our previously established physiologically detailed computer model, we show that our simulation results agree with the experimental observations that mitochondrial depolarization could induce spontaneous Ca2+ release and DADs. Furthermore, we found that it is the redox effect on RyRs that causes the DADs under the acute effect of mitochondrial depolarization, and the redox effect of ROS on reducing SERCA strength may not be sufficient to suppress DADs (Experimental findings reported that the mitochondria depolarization through the application of FCCP promotes Ca2+ waves . Further2+ waves . Also, o2+ waves . Elevatirequency . Furthernd SERCA . The dirnd SERCA , whereasnd SERCA . Thus, tess DADs .2+ release and DADs chronically, since a higher CaMKII activation level caused a greater DAD amplitude (In addition, we indeed observed an increase in CaMKII activation due to the mPTP opening , but themplitude .2+ release and DADs. Clamped ATP simulations showed that a lower cytosolic ATP level is linked to a smaller amplitude of DADs (2+ waves and DADs, our simulations and other’s experimental work suggest that ATP reduction could promote Ca2+ alternans, which is still arrhythmogenic (Similarly, we have observed a slight change in cytosolic ATP under the acute effect of mitochondrial depolarizations in our simulations . Therefo of DADs . This is of DADs . Althoughmogenic .2+ uptake has been reported to rise in heart failure (2+ alternans (2+ alternans through the Ca2+ dependent opening of mPTPs (2+ efflux in the proximity of the junctional SR played an essential role in the regulation of Ca2+ waves. Furthermore, our previous study has shown that MCU overexpression can lead to Ca2+ overload in mitochondria (2+ overload can cause the Ca2+-dependent opening of mPTP (2+ release and DADs (2+-dependent openings of mPTP, which resulted in spontaneous Ca2+ release primarily due to the direct redox effect of ROS on RyRs.Mitochondrial Ca failure  and can  failure  and Ca2+lternans . Our prelternans . And MCUof mPTPs . The prechondria . Also, t of mPTP , resulti of mPTP . Here, oand DADs . The und2+ waves and DADs under the acute effect of mitochondrial depolarization. Furthermore, the upregulation of MCU can promote DADs through Ca2+-dependent opening of mPTPs. These findings suggest that pharmacological interventions targeted at avoiding ROS buildup and MCU upregulation may provide novel therapeutics to prevent or treat cardiac arrhythmias.Mitochondrial dysfunction has been associated with increased arrhythmic risk , which c2+ handling proteins are a few examples (This detailed model coupled AP, CRUs, and mitochondria to capture excitation-contraction-metabolism coupling in ventricular myocytes. However, it has some limitations. For instance, heterogeneities in T-tubule networks and distributions of ion channels and Caexamples  that shoexamples .mPTP=1, there was virtually no DAD occurring even with αMCU=50, suggesting that the proposed mechanism of increasing MCU activity inducing DADs in this study requires a certain basal level of mPTP opening. In this model, we consider the mPTP gating kinetics only mitochondrial Ca2+ dependent. However, the ROS-induced ROS release mechanism also impacts the mPTP open probability (2+ instability. In the future, the ROS-induced ROS release will be added to our model to study the role of mitochondrial depolarization waves in the genesis of arrhythmias in diseased conditions, such as heart failure.We note that in bability , 2006, wbability . Thus, tThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.VP performed the simulations, analyzed simulation results, and drafted the manuscript. ZS prepared the figures. All the authors conceived and designed the study, interpreted the results, and edited and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the 1980s, two classes of Ca2+-binding proteins were proposed as putative Ca2+ sensors: EF-hand protein calmodulin, and the C2 domain protein synaptotagmin. In the next few decades, numerous studies determined that in the final stage of membrane fusion triggered by a micromolar boost in the level of Ca2+, the low affinity Ca2+-binding protein synaptotagmin, especially synaptotagmin 1 and 2, acts as the primary Ca2+ sensor, whereas calmodulin is unlikely to be functional due to its high Ca2+ affinity. However, in the meantime emerging evidence has revealed that calmodulin is involved in the earlier exocytotic steps prior to fusion, such as vesicle trafficking, docking and priming by acting as a high affinity Ca2+ sensor activated at submicromolar level of Ca2+. Calmodulin directly interacts with multiple regulatory proteins involved in the regulation of exocytosis, including VAMP, myosin V, Munc13, synapsin, GAP43 and Rab3, and switches on key kinases, such as type II Ca2+/calmodulin-dependent protein kinase, to phosphorylate a series of exocytosis regulators, including syntaxin, synapsin, RIM and Ca2+ channels. Moreover, calmodulin interacts with synaptotagmin through either direct binding or indirect phosphorylation. In summary, calmodulin and synaptotagmin are Ca2+ sensors that play complementary roles throughout the process of exocytosis. In this review, we discuss the complementary roles that calmodulin and synaptotagmin play as Ca2+ sensors during exocytosis.Exocytosis is a Ca Hence, Ca2+-binding proteins are required to serve as sensors to transpose Ca2+ signals in these Ca2+-dependent processes. A typical Ca2+-regulated event is exocytosis, which is a key functional cellular action, as it is responsible for a wide range processes such as the secretion of hormones from endocrine cells, the release of neurotransmitters from presynaptic neurons, the acrosome reaction during fertilization, and the delivery of plasma membrane-bound receptors , which binds Ca2+via four EF-hands. CaM is a highly conserved protein ubiquitously and abundantly expressed in eukaryotic cells , protein kinase C (PKC), and synaptotagmin (Syt).Two classes of Caic cells . The C2 2+ sensor for exocytosis. At the beginning, researchers focused on both EF-hand and C2 domain proteins because of their Ca2+ binding properties. Later studies have found that it is the C2 domain protein Syt, but not the EF-hand protein, acts as the primary Ca2+ sensor for membrane fusion. This notion was confirmed by studies in many species, including mouse  triggers the fusion of the vesicle and plasma membrane, and subsequent release of secretory cargos. This membrane fusion is driven by the SNARE complex as a core release machinery  nor t-SNAREs (target-SNAREs: syntaxin and SNAP-25) demonstrate direct interactions with Ca2+; the final process that releases secretory cargos requires a Ca2+ sensor (or Ca2+ sensors) to couple the Ca2+ signals to the SNARE-driven membrane fusion. Meanwhile, pre-fusion steps are also mediated by a [Ca2+]i slightly above resting level, although not synchronized with the boost of [Ca2+]i upon activation. Hence, Ca2+-binding proteins might also play roles in secretory vesicle lifespan before the final release.Clearly, membrane fusion, the final step, is triggered by Ca2+ in triggering the final fusion, it must require Ca2+ sensor proteins to initiate this step. In this scenario, we will highlight the process of how Syt was identified as the functional Ca2+ sensor for fusion, and why CaM was ruled out in this step.Given the critical roles of Ca2+-binding protein that consists of 148 amino acids (16.7 KDa) and coordinates four calcium ions via four EF-hands. Two of these motifs form a globular domain at the N-terminal while the other two constitute an almost symmetrical structure at the C-terminal. A flexible helical linker connects these two globular domains. In response to Ca2+ coordinating with the Ca2+-binding loops in the EF-hand motifs, a hydrophobic group in each globular domain is exposed, switching CaM to an “open” conformation that is functionally activated . This Ca2+ concentration is much lower than the boost of [Ca2+]i in most activated cells , which is above the micromolar level. If CaM serves as the Ca2+ sensor, membrane fusion would theoretically occur even in the absence of the [Ca2+]i boost. Therefore, the real Ca2+ sensor that triggers exocytotic membrane fusion should have a lower affinity to Ca2+ than CaM.Finally, 2+ sensor, Syt, is indeed responsible for the transit of Ca2+ signals to membrane fusion. Syt is a family of C2 domain-containing proteins with 17 isoforms (2+ through its tandem C2 domains (C2A and C2B) with a much lower affinity than that of CaM (Kd > 10 μM)  . Hence, syntaxin  and SNAPsyntaxin ], and thependent . This in2+ sensors for membrane fusion. In the 1990s, various studies revealed that inactivation of Syt by mutation, inhibitory peptide or knockout, largely impaired neurotransmitter release in the nematode C. elegans  and therefore does not indeed convey Ca2+ signals to release events (2+ as Ca2+ source to bypass Ca2+ channels ruled out this possibility (2+ sensor for fusion. Syt1 and Syt2 (synaptotagmin 2) are the two Syt isoforms that demonstrate the lowest Ca2+ affinity  fusion in chromaffin cells  asynchronous or spontaneous release  . The mai2+ binding, CaM may mediate the function of multiple key regulatory exocytotic proteins through direct interactions or modulation by phosphorylation via activation of some important kinases . CaMKII . This kinase was found to play a role in thyroid hormone secretion via phosphorylation of the myosin light chain  multiple regulatory proteins that are important for exocytosis. Notably, such direct interaction and indirect phosphorylation are not always isolated events. A good example is synapsin I. In order to play its role in exocytosis, synapsin I must be phosphorylated and it also serves as a substrate of CaMKII (2+-dependent binding of CaM with the head domain of synapsin 1 (via a CaM-dependent kinase and subsequent functionalization. Clearly, these two mechanisms are highly correlative. Besides synapsin, there are other proteins that are both binding partners of CaM and substrates of CaM-dependent kinase, such as VAMP and Syt. These proteins can be modulated by CaM through both mechanisms. However, it is still unclear whether these two mechanisms act synergistically or independently for each individual protein.As described above, CaM can either directly interact with or indirectly phosphorylate (f CaMKII . Interesnapsin 1 . Upon Canapsin 1 , and mobnapsin 1 , therebynapsin 1 , mediatinapsin 1 , or modunapsin 1 . In this2+ (It should be noted that, in addition to direct binding and indirect phosphorylation, CaM is also involved in other cellular processes under physiological conditions. For instance, CaM enhances ribbon replenishment and shapes filtering of synaptic transmission by functioning as an endogenous buffer of intracellular Ca2+ . It has 2+ .2+-dependent direct binding and phosphorylation via Ca2+/CaM activated-kinases. In general, a submicromolar [Ca2+]i level is required for CaM to act. Hence, CaM is considered as a high affinity Ca2+ sensor for the pre-fusion steps, while Syt is a low affinity Ca2+ sensor for the final fusion (In summary, CaM is an active mediator of exocytosis that is mainly involved in vesicle trafficking, docking and priming steps prior to membrane fusion. The major mechanism underlying this function is modulation of key regulatory proteins through Cal fusion .2+-binding proteins. CaM, the EF-hand protein, can interact with Syt, the C2 domain protein. Hence, to some extent, the function of the Ca2+ sensor in membrane fusion might also be modulated by CaM. via a neuroxin-binding region close to the C-terminal. However, the physiological function of this interaction remained unknown until recent report that CaM-Syt7 binding is essential for the Syt7-mediated refilling of the RRP during synaptic depression (2+ sensing modules in the CaM-Syt7 complex.Finally, it is worth paying some attention to the direct interplay between the two major classes of Capression  and the pression . These f2+-bound CaM (2+-dependent interaction with t-SNAREs (via either direct binding or indirect phosphorylation. It is still unknown whether the indirect phosphorylation is dependent on the direct binding, as for synapsin. Future studies are required to address this question.In addition to direct binding, it has been reported that Syt can be phosphorylated by CaMKII, which is activated by Caound CaM . This pht-SNAREs . In ligh2+/CaM suppresses the expression of Syt2 in cortical neurons via an unknown mechanism (in vitro liposome fusion assay, CaM was also found to inhibit, rather than facilitate, membrane fusion in the absence of Syt, with these inhibitory effects being abolished in the presence of Syt (CaM and Syt can also inhibit each other. It has been reported that Caechanism . In an ie of Syt . These s2+ signals and consequently triggers the final fusion events. Although not the Ca2+ sensor for fusion, CaM might act as a putative high affinity Ca2+ sensor that is switched on at low [Ca2+]i, which is close to resting [Ca2+]i, thereby working in temporal disassociation from the [Ca2+]i boost or spatially distant to the Ca2+ micro/nano domain. Hence, it mainly mediates early exocytotic steps prior to membrane fusion, such as vesicle trafficking, docking and priming, via direct interactions with and/or indirect phosphorylation of key regulatory exocytotic proteins.In conclusion, during exocytosis, Syt, but not CaM, senses Ca2+-binding proteins CaM during exocytosis, several key questions need to be addressed in future studies. Does CaM play a secondary role in final membrane fusion? What is the impact of the CaM-Syt interaction in exocytosis? Are any interaction partners of CaM or substrates of CaM-dependent kinases still to be identified? Addressing these questions will enhance our understanding of the entire process of exocytosis and further illuminate how exocytosis is precisely regulated by calcium ions and a plethora of proteins.To further understand the roles of CaRX, HM, JY, JX, ZH, and HL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SOCE-dependent cytosolic Ca2+ signals are affected by mitochondrial Ca2+ transport through several competing mechanisms. However, how these mechanisms interact in shaping Ca2+ dynamics and regulating Ca2+-dependent functions remains unclear. In a recent issue, Yoast et al. shed light on these questions by defining multiple roles of the mitochondrial Ca2+ uniporter in regulating SOCE, Ca2+ dynamics, transcription, and lymphocyte activation.Store-operated Ca Depletion of ER Ca2+ triggers the activation of Ca2+ release–activated Ca2+ (CRAC) channels in the plasma membrane allowing Ca2+ influx into the cell (2+ entry (SOCE) serves to refill ER Ca2+ stores, shape cytosolic Ca2+ signaling, and regulate numerous Ca2+-dependent functions. Once activated, CRAC channels undergo Ca2+-dependent inactivation (CDI), which limits SOCE through a negative feedback mechanism . However, the net effect of mitochondria on the Ca2+ signal depends on several additional factors, including the ability of mitochondria to release accumulated Ca2+ back into the cytosol, regulate Ca2+ dynamics within the ER, and generate ATP in a Ca2+-dependent manner. The complex problem of how these mitochondria-dependent mechanisms work together to shape SOCE-mediated Ca2+ signaling is the focus of the study by Yoast et al.   , 4, in v changes . Unexpecansients . A simil2+ signaling. First, using whole-cell patch-clamp recordings, they found that MCU KO promoted inactivation of CRAC currents, consistent with previous reports that mitochondrial Ca2+ buffering reduces CDI of CRAC channels  and electron transport inhibitors  to block mitochondrial Ca2+ buffering by inducing mitochondrial depolarization and thereby dissipating the driving force for mitochondrial Ca2+ uptake  and its control of immune function 2+ signalne cells . It is ihis work  and earlhis work .Figure 12+ signaling, including buffering cytosolic Ca2+, reducing CDI of CRAC channels, and regulating ER Ca2+ store replenishment  and nonexcitable cells , and determining how the cell type–specific molecular composition of MCU complexes and CRAC channels and their subcellular localizations are optimized for the control of distinct functions , are important questions to examine. Future research will help address these and many other critical questions inspired by this article.As with any insightful study that moves the field forward, the article by Yoast The authors declare that they have no conflicts of interest with the contents of this article.