Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Gen Physiol
2018 Jul 02;1507:1035-1043. doi: 10.1085/jgp.201812015.
Show Gene links
Show Anatomy links
Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes.
Tsai CW
,
Tsai MF
.
???displayArticle.abstract???
The mitochondrial calcium uniporter is a multisubunit Ca2+ channel that mediates mitochondrial Ca2+ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca2+-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca2+ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca2+ > Sr2+ > Ba2+, Mn2+, and Mg2+ Patch clamp recordings further reveal macroscopic and single-channel Ca2+ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca2+-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.
???displayArticle.pubmedLink???
29891485
???displayArticle.pmcLink???PMC6028504 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. Subunit assembly in the uniporter complex. The red dashed line indicates how MCU and EMRE are fused to produce the hME tandem construct.
Figure 2. Expression of hME in Xenopus oocytes. The Western blot image shows expression of WT hME and two nonfunctional mutants. Each lane represents an independent repeat. Oocytes were injected with 12 ng cRNA and harvested after 3–4 d of incubation. 25 oocytes were used for each membrane preparation. MW, molecular weight.
Figure 3. Uniporter-induced ICACC. (A) TEVC recordings of hME-expressing oocytes. Currents were recorded using repeated voltage ramps (inset). Left: I–V relations. (1) 2 mM Ca2+. (2) 20 mM Ca2+. (3) 20 mM Ca2+ + 1 µM Ru360. Right: Currents at 80 mV. (B) ICACC in hME-expressing or uninjected (con) oocytes. The bar chart compares currents (80 mV) induced by 2 mM (gray) and 20 mM (red) extracellular Ca2+. Numbers indicate independent repeats. Data are presented as mean ± SEM.
Figure 4. Isolation of IMCU by applying intracellular Ca2+ chelators. (A) Currents induced by 20 mM extracellular Ca2+ in the presence (red) or absence (black) of 1 µM Ru360. Oocytes were preinjected with 5 nmol EGTA and were constantly exposed to 0.5 mM niflumic acid during recordings. (B) IMCU, obtained by subtracting Ru360-insentive currents (red in A) from total currents (black in A). (C) Currents in uninjected control. Adding Ru360 (red trace) does not affect currents. All traces were digitally filtered at 100 Hz.
Figure 5. IMCU in low ICACC oocytes. (A) Currents induced by: a, 2 mM Ca2+; b, 20 mM Ca2+; and c, 20 mM Ca2+ + 1 µM Ru360. (B) IMCU, obtained by subtracting c from b. (C) Currents from oocytes expressing G353W-hME. Color code is the same as in A.
Figure 6. Ca2+ dose response of IMCU. (A) I–V curves of IMCU in various external [Ca2+]. (B) A dose–response plot. Currents (measured at −120 mV) induced by 2, 5, 10, and 50 mM Ca2+ were normalized to currents induced by 20 mM Ca2+. A Michaelis-Menten equation was used for data fit (red curve). Each data point represents five to eight repeats. Data are presented as mean ± SEM.
Figure 7. Ru360 inhibition of IMCU. (A) Left: I–V relation of IMCU in the presence of 20 mM Ca2+ and various concentrations of Ru360. Right: currents at −120 mV plotted in a time course. (B) A Ru360 dose-response curve. Data fitting uses a standard single-site binding model (red curve). Each data point represents the mean of at least five independent repeats. Data are presented as mean ± SEM. (C) Current (−120 mV) recovery upon Ru360 (500 nM) removal follows a single exponential curve (red). The voltage protocol is the same as in Fig. 6.
Figure 8. Divalent cation conductivity. (A) Currents (−120 mV) of hME elicited by 20 mM of various divalent cations. Black bars indicate Ca2+ concentrations in mM. The voltage protocol is the same as in Fig. 6. (B) A comparison of divalent cation transport. All currents were induced by 20 mM divalent cations. Data are presented as mean ± SEM.
Figure 9. Mn2+ inhibition of IMCU. (A) Inhibition of IMCU (−120 mV) by Mn2+ at indicated concentrations in mM. (B) A dose response of Mn2+ inhibition. Curve fitting (red curve) assumes single-site binding. Data are presented as mean ± SEM.
Figure 10. Patch clamp outside-out recordings of hME. (A and B) Macroscopic recordings with currents >5 pA. (C and D) Recordings with discernible single-channel events. Red curves: Single exponential fit of current recovery after Ru360 removal. Blue bars: 200 nM Ru360. Bath contains 100 mM Ca2+. Pipette solutions contain 5 mM EGTA and 5 mM EDTA to reduce free Ca2+ below 1 nM to inhibit ICACC. Voltage was clamped at −80 mV for all recordings.
Baughman,
Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.
2011, Pubmed
Baughman,
Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.
2011,
Pubmed
Bernardi,
Mitochondrial transport of cations: channels, exchangers, and permeability transition.
1999,
Pubmed
Bragadin,
Kinetics of Ca2+ carrier in rat liver mitochondria.
1979,
Pubmed
Cao,
Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter.
2017,
Pubmed
Cibulsky,
Block by ruthenium red of cloned neuronal voltage-gated calcium channels.
1999,
Pubmed
,
Xenbase
Csordás,
MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca²⁺ uniporter.
2013,
Pubmed
De Stefani,
A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter.
2011,
Pubmed
Gunter,
Mitochondrial calcium transport: mechanisms and functions.
2000,
Pubmed
Hartzell,
Activation of different Cl currents in Xenopus oocytes by Ca liberated from stores and by capacitative Ca influx.
1996,
Pubmed
,
Xenbase
Jegla,
A novel subunit for shal K+ channels radically alters activation and inactivation.
1997,
Pubmed
Jorgensen,
Annexins from Ehrlich ascites cells inhibit the calcium-activated chloride current in Xenopus laevis oocytes.
1997,
Pubmed
,
Xenbase
Kamer,
The molecular era of the mitochondrial calcium uniporter.
2015,
Pubmed
Kamer,
High-affinity cooperative Ca2+ binding by MICU1-MICU2 serves as an on-off switch for the uniporter.
2017,
Pubmed
Kamsteeg,
Detection of aquaporin-2 in the plasma membranes of oocytes: a novel isolation method with improved yield and purity.
2001,
Pubmed
,
Xenbase
Kirichok,
The mitochondrial calcium uniporter is a highly selective ion channel.
2004,
Pubmed
Kovács-Bogdán,
Reconstitution of the mitochondrial calcium uniporter in yeast.
2014,
Pubmed
Kunzelmann,
The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl- conductance of Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Kwong,
The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart.
2015,
Pubmed
Li,
Expression and preliminary characterization of human MICU2.
2016,
Pubmed
Liu,
MICU1 Serves as a Molecular Gatekeeper to Prevent In Vivo Mitochondrial Calcium Overload.
2016,
Pubmed
Logan,
Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling.
2014,
Pubmed
Luongo,
The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition.
2015,
Pubmed
Ma,
Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle.
1993,
Pubmed
Mallilankaraman,
MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake that regulates cell survival.
2012,
Pubmed
Matlib,
Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes.
1998,
Pubmed
Oxenoid,
Architecture of the mitochondrial calcium uniporter.
2016,
Pubmed
Patron,
MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity.
2014,
Pubmed
Penna,
The MCU complex in cell death.
2018,
Pubmed
Petrungaro,
The Ca(2+)-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca(2+) Uptake.
2015,
Pubmed
Rigoni,
Ruthenium red inhibits the mitochondrial Ca2+ uptake in intact bovine spermatozoa and increases the cytosolic Ca2+ concentration.
1986,
Pubmed
Rizzuto,
Mitochondria as sensors and regulators of calcium signalling.
2012,
Pubmed
Sancak,
EMRE is an essential component of the mitochondrial calcium uniporter complex.
2013,
Pubmed
Sather,
Permeation and selectivity in calcium channels.
2003,
Pubmed
Tsai,
Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.
2016,
Pubmed
Vais,
EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter.
2016,
Pubmed
Wall,
Isolation of plasma membrane complexes from Xenopus oocytes.
1989,
Pubmed
,
Xenbase
Wang,
Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake.
2014,
Pubmed
White,
Niflumic and flufenamic acids are potent reversible blockers of Ca2(+)-activated Cl- channels in Xenopus oocytes.
1990,
Pubmed
,
Xenbase
Wu,
Single channel recording of a mitochondrial calcium uniporter.
2018,
Pubmed
Yamamoto,
Analysis of the structure and function of EMRE in a yeast expression system.
2016,
Pubmed
Yao,
Calcium current activated by depletion of calcium stores in Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Ying,
Inhibition of mitochondrial calcium ion transport by an oxo-bridged dinuclear ruthenium ammine complex.
1991,
Pubmed