The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit

Abstract

Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red-sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino-acid substitutions in the pore region and indeed MCUb does not form a calcium-permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant-negative effect, reducing the [Ca 2 þ ] mt increases evoked by agonist stimulation. Accordingly, in vitro co-expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant-negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

“…6). The size of the MCU monomer and oligomer observed here was consistent with our observation using wheat germ lysates expressing MCU (63). The magnitude of this higher order complex increased following 15 min of Phe stimulation, which was blocked by either Mito-TEMPO or Pyk2-knockdown (Fig.…”

“…S2). The increase in [Ca 2+ ] mt observed in response to an elevation in cytosolic Ca 2+ induced by TG was almost abolished by expression of a dominant-negative pore-forming subunit of MCU (MCUB) (63), confirming the MCU dependence of this mitochondrial Ca 2+ uptake ( Fig. 1D-G).…”

“…We next addressed the mechanism by which Pyk2-dependent MCU phosphorylation activates MCU-dependent mitochondrial Ca 2+ uptake. A single MCU protein contains two transmembrane domains and MCU needs to be oligomerized to form channels (14,63). Therefore, our next hypothesis is that Pyk2-dependent MCU phosphorylation enhances MCU oligomerization (i.e., channel pore formation).…”

Reduced mitochondrial Ca2+ transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase

“…The MCU-flag/pcDNA expression construct is described in De Stefani et al 12 The Parkin full-length expression construct is described in Cali et al 51 The MCU-flag adenovirus production is described in Raffaello et al 14 Immunofluorescence. Control and mutated fibroblasts or wt and mutated cybrids were starved (KRB for 4 h), treated with chloroquine (50 μM for 1 h), Kaempferol (20 μM for 24 or 48 h) or SB202190 (20 μM for 24 or 48 h) and transfected with Parkin, MCU or empty vector as control, as indicated.…”

“…MCU was identified in 2011, 12,13 and in the following years, molecular insight on its complex regulatory mechanism was obtained. The pore region is composed of MCU, its isoform MCUb 14 and essential MCU regulator (EMRE). 15 The channel is gated by the Ca 2+ -sensitive proteins mitochondrial Ca 2+ uptake 1 (MICU1) and MICU2 [16][17][18][19] and further regulated by the SLC25A23 protein.…”