Tsai CW and Tsai MF (2018), Electrical recordings of the mitochondrial calc...

XB-ART-55031

J Gen Physiol 2018 Jul 02;1507:1035-1043. doi: 10.1085/jgp.201812015.

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Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes.

Tsai CW , Tsai MF .

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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.

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GO keywords: oxidative phosphorylation

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	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.

References [+] :

Baughman, Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. 2011, Pubmed