Calcium influx through the mitochondrial calcium uniporter holocomplex, MCUcx

Abstract

Ca²⁺ flux into the mitochondrial matrix through the MCU holocomplex (MCU_cx) has recently been measured quantitatively and with milliseconds resolution for the first time under physiological conditions in both heart and skeletal muscle. Additionally, the dynamic levels of Ca²⁺ in the mitochondrial matrix ([Ca²⁺]_m) of cardiomyocytes were measured as it was controlled by the balance between influx of Ca²⁺ into the mitochondrial matrix through MCU_cx and efflux through the mitochondrial Na⁺ / Ca²⁺ exchanger (NCLX). Under these conditions [Ca²⁺]_m was shown to regulate ATP production by the mitochondria at only a few critical sites. Additional functions attributed to [Ca²⁺]_m continue to be reported in the literature. Here we review the new findings attributed to MCU_cx function and provide a framework for understanding and investigating mitochondrial Ca²⁺ influx features, many of which remain controversial. The properties and functions of the MCU_cx subunits that constitute the holocomplex are challenging to tease apart. Such distinct subunits include EMRE, MCUR1, MICUx (i.e. MICU1, MICU2, MICU3), and the pore-forming subunits (MCU_pore). Currently, the specific set of functions of each subunit remains non-quantitative and controversial. The more contentious issues are discussed in the context of the newly measured native MCU_cx Ca²⁺ flux from heart and skeletal muscle. These MCU_cx Ca²⁺ flux measurements have been shown to be a highly-regulated, tissue-specific with femto-Siemens Ca²⁺ conductances and with distinct extramitochondrial Ca²⁺ ([Ca²⁺]_i) dependencies. These data from cardiac and skeletal muscle mitochondria have been examined quantitatively for their threshold [Ca²⁺]_i levels and for hypothesized gatekeeping function and are discussed in the context of model cell (e.g. HeLa, MEF, HEK293, COS7 cells) measurements. Our new findings on MCU_cx dependent matrix [Ca²⁺]_m signaling provide a quantitative basis for on-going and new investigations of the roles of MCU_cx in cardiac function ranging from metabolic fuel selection, capillary blood-flow control and the pathological activation of the mitochondrial permeability transition pore (mPTP). Additionally, this review presents the use of advanced new methods that can be readily adapted by any investigator to enable them to carry out quantitative Ca²⁺ measurements in mitochondria while controlling the inner mitochondrial membrane potential, ΔΨ_m.

Keywords: Heart; Mitochondrial Ca2+ signaling; Mitochondrial Na+/Ca2+ exchanger (NCLX); Mitochondrial calcium uniporter complex (MCUcx); Mitochondrial permeability transition pore (mPTP); Skeletal muscle.

Figure 1.. MCU_cx flux and its regulation by [Ca²⁺]_i in heart.

a. Measurements of the MCU_cx Ca²⁺ influx (J_mcu) in heart mitochondria (units are scaled to liter of cytosol). J_mcu (μM s⁻¹) is plotted as a function of measured extramitochondrial, [Ca²⁺]_i. Each of these measurements of J_mcu were carried out along with measurements of [Ca²⁺]_i, [Ca²⁺]_m, and ΔΨ_m. The inset (top) is a zoomed-in view of the region of the plot between 0 and 3 μM [Ca²⁺]_i. Linear least-squares fit to the filled circles is shown (slope = 1.2, going through the origin at 0, 0). b. MCU_cx conductance (G) for each of 63 experiments shown in a, normalized to the minimal conductance (G_min) of each dataset (G/G_min). G/G_min is plotted as a function of [Ca²⁺]_i. Inset (top) is a zoomed-in view of the region between 0 and 3 μM [Ca²⁺]_i. Linear least- squares fit line to the filled circles is shown (slope = 6.1, going through the origin at 0,0). c. Number of open MCU_cx channels per mitochondrion plotted as a function of [Ca²⁺]_i. Taken from b after division by the number of mitochondria per liter cytosol and dividing by the [Ca²⁺]_i-dependent unitary conductance of MCU_cx (For additional technical details see [3, 7, 9]). Linear least-squares fit to the filled circles is shown (slope = 0.116, intercept = 7.48). Panels a-c are taken with publisher permission from Wescott et. al., Nature Metabolism, 2019 [3].

Figure 2.. MCU_cx and its regulation by [Ca²⁺]_i in heart and skeletal muscle.

a. Measurement of the MCU_cx Ca²⁺ influx (J_MCU-cx) in cardiac mitochondria (green circles), skeletal muscle (black circles), and skeletal muscle with Ru360 (5 μM, red circles). For convenience on the graphs, J_mcu is used to represent J_MCU-cx . Thus J_mcu is plotted as a function of measured extramitochondrial [Ca²⁺]_i. Each of these measurements of J_mcu were carried out along with measurements of [Ca²⁺]_i, [Ca²⁺]_m, and ΔΨ_m. Linear least-squares fit to the heart mitochondria data is shown (slope = 0.015). b. Zoomed-in view of region between 0 and 3 μM of [Ca²⁺]_i. from a. Black and green filled circles are from [3]. Overlaid dashed lines intercept the vertical axis at the [Ca²⁺]_i levels where conduction thresholds of the MCU_cx were proposed to occur. The levels of [Ca²⁺]_i at which each threshold was proposed to be are taken from the indicated studies. The red filled circle is based on the threshold value from references [11-15], the purple from[16-18], the green from reference [19] c. Relative number of open MCU_cx channels per mitochondrion plotted as a function of [Ca²⁺]_i (For additional technical details see [3, 7, 9]). Linear least-squares fit to the heart mitochondria data is shown (slope = 0.051, intercept = 3.3). Skeletal muscle MCU_cx data was fitted to a modified Hill equation yielding a K_0.5 of 7.9 μM and a Hill coefficient of 2.95. Panels a-c are taken with publisher permission from Wescott et. al., Nature Metabolism, 2019 [3].