Role of MCU for mitochondrial pH gradient

In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca influx, which triggers insulin exocytosis. The mitochondrial Ca uniporter (MCU) mediates Ca uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilised clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCUsilenced cells. In contrast, nutrient-induced hyperpolarisation of the electrical gradient was not altered. In permeabilised cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca. Suppression of the putative Ca/H antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca-induced matrix acidification. These results demonstrate that MCU-mediated Ca -----------------------------------------------------------uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells. http://www.jbc.org/cgi/doi/10.1074/jbc.M114.632547 The latest version is at JBC Papers in Press. Published on December 29, 2014 as Manuscript M114.632547 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Role of MCU for mitochondrial pH gradient 2 Pancreatic β-cells maintain blood glucose homeostasis by adapting insulin secretion to the changes in circulating nutrients. A major signaling molecule in this metabolism-secretion coupling linking nutrient metabolism to insulin secretion is cytosolic ATP most of which is synthesized from oxidative phosphorylation. Mitochondrial ATP synthesis is driven by the electrical (ΔΨmito, membrane potential) and chemical (ΔpHmito 1 ) gradients across the mitochondrial inner membrane. These gradients are established as a result of electron transport and the associated export of protons mediated by the respiratory chain. Reducing equivalents in mitochondrial matrix are mainly produced by the tricarboxylic acid (TCA) cycle and mitochondrial metabolite shuttles. Thus, the metabolic status of the β-cell mitochondria critically controls ATP synthesis and insulin secretory activity ( ). Accumulating evidence suggests that defective mitochondrial function results in impaired glucose-stimulated insulin secretion (GSIS) and may contribute to the development of type 2 diabetes (2-5). The matrix Ca concentration ([Ca]mito 1 ) is a key activator of mitochondrial metabolic function ( ,6,7). The [Ca]mito 8 activates several matrix enzymes including α-ketoglutarate dehydrogenase in the TCA cycle ( ). The ATP synthase is also directly activated by a rise in [Ca]mito 9 ( ). In pancreatic β-cells [Ca]mito 10 is strictly required for ATP synthase-dependent respiration stimulated by glucose ( ). Given its importance, mitochondrial Ca uptake has been a research focus for decades, starting with the functional characterization in isolated mitochondria. Nevertheless, it took 50 years to elucidate the molecular identity of the mitochondrial Ca 11 uniporter (MCU) ( ,12). Mitochondrial Ca uptake through MCU is regulated by a number of recently discovered proteins, including mitochondrial Ca 13-15 uptake 1 and 2 (MICU1/2) ( ), mitochondrial Ca 16 uniporter regulator 1 (MCUR1) ( ), and essential MCU regulator (EMRE) (17). Especially MICU1/2 negatively regulate MCU activity under resting cytosolic Ca ([Ca] i). At stimulating [Ca] i (> 10 μM), however, MICU1 activates MCU activity, implying that the regulatory subunits of the MCU complex modulate mitochondrial Ca loads of ΔΨmito-driven Ca 13 uptake without perturbing the important signal propagation from ER to mitochondria ( ,18,19). Mitochondrial Ca homeostasis is maintained by balanced Ca influx and efflux. Mitochondrial Ca export is mediated by antiporters exchanging Ca for H or Na 20 ( ). Two mitochondrial antiporters promoting Ca efflux have been identified: The leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) and the mitochondrial sodium calcium exchanger (NCLX). LETM1, which is defective in Wolf-Hirschhorn syndrome (WHS), works as a K/H 21 exchanger in yeast mitochondria ( ) or mammalian ER (22). LETM1 was also shown to mediate Ca/H exchange in mitochondria with a [Ca]mito 23 dependent biphasic mode ( ). NCLX was confirmed as an electrogenic Na/Ca antiporter (exchanging 3 or 4 Na per Ca 24 ) ( ) . Inhibition of NCLX in pancreatic β-cells increases [Ca]mito 25-28 , accelerates mitochondrial oxidative metabolism and GSIS ( ). In addition to [Ca]mito, the matrix pH has been identified as a regulator of mitochondrial energy metabolism in β-cells. In contrast to other cell types, pancreatic β-cells have acidic pHmito 29 under resting conditions. Nutrient stimulation causes matrix alkalinization without any marked cytosolic pH change ( ). Preventing the resulting nutrient-induced increase of the ΔpHmito changes using ionophores abrogated proton-coupled mitochondrial ion/metabolite transport, ATP synthesis, and GSIS regardless of elevated ΔΨmito 29-31 ( ). Therefore, pathogenic conditions causing a reduction of ΔpHmito Several recent reports demonstrate the functional role of MCU in pancreatic β-cells ( may seriously deteriorate ATP generation and insulin secretion in pancreatic