1. Biochemistry and Chemical Biology
  2. Cell Biology
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MCU encodes the pore conducting mitochondrial calcium currents

  1. Dipayan Chaudhuri
  2. Yasemin Sancak
  3. Vamsi K Mootha
  4. David E Clapham  Is a corresponding author
  1. Massachusetts General Hospital, United States
  2. Howard Hughes Medical Institute, Boston Children’s Hospital, United States
  3. Harvard Medical School, United States
Research Article
Cite this article as: eLife 2013;2:e00704 doi: 10.7554/eLife.00704
2 figures

Figures

MCU expression recapitulates IMiCa.

(A) A HEK-293T cell mitoplast under differential interference contrast (far left), showing a typical figure-eight shape. The lobe bounded only by the mitochondrial inner membrane appears less dense (white arrow) than the lobe also bounded by the outer membrane (black arrow). Matrix-targeted mCherry demonstrates that the inner membrane forms a surface on both lobes (middle left). GFP-tagged mitofusin-1 (an outer membrane GTPase, middle right) reveals the outer membrane partially encapsulating only one lobe. Far right: merged image. (B) Western blots demonstrate reduced MCU expression following short-hairpin RNA-mediated knockdown of MCU but not GFP (control). ATP5B is a loading control. (C) Exemplar current traces demonstrate endogenous IMiCa in control knockdown (black trace), which is largely blocked by 100 nM RuR (red trace). Voltage ramps from −160 to +80 mV for 750 ms were delivered every 6 s from a holding potential of 0 mV. (D) Significantly reduced IMiCa after MCU knockdown. (E) Summary data, n = 6–10 per condition, error bars report SEM.

https://doi.org/10.7554/eLife.00704.003
MCU mutants alter IMiCa sensitivity to RuR.

(A) Confocal imaging of HEK-293T cells for FLAG-tagged MCU (left) and cytochrome C oxidase I (a mitochondrial marker, center). The merged image (right) shows colocalization. (B) As in (A) but for MCU-259A-FLAG. (C) Immunoblot analysis of proteinase K digestion after wild-type MCU-FLAG-expressing HEK-293T mitochondrial fractions are digitonin-permeabilized at the specified concentrations, confirming mitochondrial targeting. (D) Proteinase K digestion analysis for MCU-S259A-FLAG mutant. (E) Significant increase in IMiCa after MCU-FLAG overexpression. (F) Loss of RuR block in S259A mutant. (G) Summary data, n = 5–9 per condition.

https://doi.org/10.7554/eLife.00704.004

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