The mechanism of MICU-dependent gating of the mitochondrial Ca2+uniporter

  1. Vivek Garg
  2. Junji Suzuki
  3. Ishan Paranjpe
  4. Tiffany Unsulangi
  5. Liron Boyman
  6. Lorin S Milescu
  7. W Jonathan Lederer
  8. Yuriy Kirichok

  • Curated by eLife

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    Evaluation Summary:

    This paper examines the roles and mechanisms of how subunits of the mitochondrial calcium uniporter complex (MCUcx) 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 preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

Ca 2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCU cx ), a Ca 2+ -selective channel composed of five subunit types. Two MCU cx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca 2+ -regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca 2+ and Na + fluxes via MCU cx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCU cx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCU cx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca 2+ as has been widely reported. Instead, MICUs potentiate activity of MCU cx as extramitochondrial Ca 2+ is elevated. MICUs achieve this by modifying the gating properties of MCU cx allowing it to spend more time in the open state.

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  1. Version published to 10.7554/elife.69312 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    We would like to thank the reviewer for commenting on the strength of the approach. We addressed the question regarding the effect of Ca2+ and Mg2+ on the association of MICUs with MCUcx in Essential Revisions points 1 and 2 (below).

    It remains an open question why MICU1-/- mitochondria have increased mitochondrial Ca2+ uptake in the low [Ca2+]i range. The answer to this question may not be straightforward since MICUs might have effects outside the MCUcx. Extensive future studies will be required to answer this question definitively.

    Reviewer #2 (Public Review):

    We would like to thank the reviewer for the encouraging comments. Potential biphasic effect of Ca2+ on MICUs was addressed in Essential Revisions point 1 (below).

    Reviewer #3 (Public Review):

    Thank you very much for commenting on the strengths of the manuscript. We agree that the data presented will foster further experiments and discussions regarding MICUs and will eventually reveal their true mechanism of action.

    Essential Revisions 1)

    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+], 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 (Figure 7) 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.

    “Why does knocking out MICU1 tends to increase the net mitochondrial 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 (see Gottschalk et al., 2019; Tomar et al., 2019; Tufi et al., 2019) -- 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 (Kamer et al., 2017) (Figs. 1E, 1G, 2F, 2H, 2J and 3H). 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.

    We agree with the reviewers comment that Wang et al 2020b showed the possibility of the occlusion in the native dimeric form of the MCUcx. 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 paragraph discussing MCUcx structures to reflect these changes.

    Although 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.

    Essential Revisions 2)

    We have added a set of new electrophysiological experiments to address the effect of Mg2+ on Ca2+ currents. 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 would also like to reemphasize that in this study we did not center our investigation on the net mitochondrial Ca2+uptake, but focus specifically on MCUcx activity. The increase in net mitochondrial Ca2+ uptake in MICU1-/- vs WT was observed both in the presence (Csordas et al., 2013) or absence of Mg2+ (Mallilankaraman et al., 2012). Thus, the putative occlusion of the MCU pore by MICUs, if it exists, would be a Mg2+-independent phenomenon.

    With these new results, we will revise the results and the discussion sections. We will highlight the impact that physiological Mg2+ block has, in limiting MCUcx flux at low Ca2+. We will also emphasize the importance of reevaluating the structure of MCU holocomplex in the Mg2+ bound conformation. We thank the reviewers for prompting this addition.

    Per reviewers’ request, we will clearly indicate in the text that the use of EDTA removes not only Ca2+ but also Mg2+.

  3. eLife

    Evaluation Summary:

    This paper examines the roles and mechanisms of how subunits of the mitochondrial calcium uniporter complex (MCUcx) 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 preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This work challenges current models for the regulation of Ca2+ uptake by the MCU channel complex in mitochondria, an important mechanism that matches the rate of ATP generation to cellular metabolic needs. Current models posit that the MICU1 subunit blocks the MCU pore at low cytosolic [Ca2+]i, and that this block is relieved by [Ca2+]i elevation, causing a steep uptake of Ca2+ once a threshold is released. This paper challenges this view, based on a series of cutting-edge patch-clamp measurements of MCU activity in mitoplasts derived from wild type and CRISPR MICU1 knockout mitochondria. This is a powerful approach as it is directly monitors MCU activity and allows precise control of the ionic environment. The mitoplast system is well validated, both in terms of the expression and knockout of MCU, MICU1/2, EMRE subunits. Using this approach, the authors have succeeded in directly addressing the function of MICU1 in regulating the activity of MCU, as well as previous claims that MCU can act as a Ca2+ release channel and that MICU1 selectively inhibits the entry of Mn2+, which can be toxic.

    The results show that the characteristic inward rectification of current through MCU as well as Mn2+ permeation are independent of MICU, and that MCU conducts Ca2+ only into mitochondria and does not act as a Ca2+-activated release channel. Most importantly, they find that in the absence of divalent cations, which allows Na+ to permeate the channel, MICU1 does not affect current amplitude, arguing strongly against its role as a blocker at low Ca2+ levels. Rather, it acts to potentiate channel activity at higher levels of Ca2+, which is dependent on Ca2+ binding to the MICU1 EF hands. By measuring single-channel currents, they show that MICU1 potentiates flux by increasing the open probability of the channel.

    These studies do much to clarify the mechanism of MICU1 modulation of Ca2+ uptake, yet there remain some open questions. The evidence against MICU1 as a pore blocker was collected in the absence of Ca2+ and Mg2+, which may affect the association of MICU1 with the channel. Ca2+ uptake by intact mitochondria is suppressed by MICU1 at low Ca2+ levels, and it remains to be shown what mechanisms account for this if MICU1 does not plug the channel.

  5. eLife

    Reviewer #2 (Public Review):

    The uptake of Ca2+ into mitochondria through the mitochondrial Ca2+ uniporter complex (MCUcx) is important for ATP production and cellular bioenergetics. The MCUcx consists of five subunits - MCU and EMRE which form the channel pore in the inner mitochondrial membrane, and MICU1-3 subunits which lie in the intermembrane space. While several previous studies have examined the roles of MICU1-3 in regulating Ca2+ uptake via MCUcx, there are ambiguities regarding their precise mechanisms. This study seeks to clarify the impact of MICU1-3 on MCUcx and the mechanisms of action of these proteins. The authors generated cell lines featuring knockout of individual MCUcx subunits; measure mitochondrial Ca2+ uptake in intact cells and isolated mitochondria; and conduct patch-clamp recordings of macroscopic and single channel MCUcx currents using Ca2+ and Na+, respectively, as charge carrier. The authors find that MICU1/MICU2 heterodimer (or MICU1 homodimer in MICU2 knockouts) potentiates MCUcx open probability in a Ca2+-dependent manner. They further conclude from their results that MICU1 does not occlude the MCUcx pore at low [Ca2+]i as has been previously reported.

    Overall, the challenging experiments in this work are technically well done and the results produce some new insights into MICU1 regulation of MCUcx. The conclusion that MICU1/MICU2 heterodimer potentiates MCUcx activity in a high [Ca2+]i-dependent manner is well supported by the data (Ca2+-dependence of Ca2+ conductance in mitochondria isolated from WT and MICU1-/- cells; single channel recordings of Ca2+ and Na+ currents in WT and MICU-/- cells).

    A fair amount of effort is put in the paper to argue that the results discount a previously proposed model that MICU1 occludes the MCUcx at resting Ca2+ levels in cells. In my view, the data supporting this conclusion is not compelling. The main issue is that the conditions which the authors use to make this argument are essentially 0 [Ca2+]i. This is different from the case in a cell where resting Ca2+ is ~100 nM. It is possible that Ca2+ has a biphasic effect on the action of MICU1/MICU2 heterodimers - inhibition of MCUcx activity at 'low' [Ca2+] ({less than or equal to} 3 microM) and potentiation of activity at high [Ca2+]i ({greater than or equal to} 10 microM). MICU1/MICU2 heterodimers have several EF hands between them and there is ample precedent for Ca2+ binding proteins with multiple EF-hands, such as calmodulin, bifurcating Ca2+ signals and to have functionally opposite effects on a target protein.

  6. eLife

    Reviewer #3 (Public Review):

    In this study, Gard et.al performed rigorous electrophysiology analysis of MCU channel complex in intact isolated mitochondria using whole mitoplast patch clamp method. Their results suggest that the MCU/EMRE channel complex (which form the channel pore) is not blocked by MICUs in the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCU complex by increasing the channel open probability when extramitochondrial Ca2+ is elevated. This study indeed provides very different views on multiple aspects of MCU complex functions. Particularly, the finding of non-inhibitory role of MICU1 is opposite to the current view of MICU1 blocking of MCU at low Ca2+ based on classical mitochondria Ca2+ uptake assays. This finding is also different from the recent structural studies of MCU/EMRE/MICU1/MICU2 holocomplex which showed that MICU1/MICU2 can bind and block the external entrance of the MCU/EMRE channel pore in the absence of Ca2+. Despite these discrepancies, this study provides quantitative analysis of MCU activity by using a highly challenging mitoplast patch clamp technique that very few labs are capable of and gives some novel insights into MCU complex activity and regulation. How to reconcile the different views of MICUs' regulation of MCU will be an interesting and exciting subject in MCU field that warrants further studies and the findings of this study will certainly stimulate this effort.

  7. Version published to 10.1101/2020.04.04.025833v3 on bioRxiv
  8. Version published to 10.1101/2020.04.04.025833v2 on bioRxiv
  9. Version published to 10.1101/2020.04.04.025833v1 on bioRxiv