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Mitochondrial Ca2+ uptake is central to oxidative metabolism and cell death signaling. The first clues to its molecular mechanism have emerged from the recent identification of the mitochondrial Ca2+ uniporter's pore forming protein (MCU) as well as its regulators. Among the regulators, MICU1 shows striking co-expression and co-evolution with MCU. MICU1 has been demonstrated to be a Ca2+-sensing protein, which both sets a threshold for low Ca2+ concentration while it assures cooperative activation during high Ca2+ rises. Mitochondrial Ca2+ uptake shows tissue specific differences and interestingly, mRNA level for MICUs and MCUs also displays tissue specificity. We set out to investigate if the stoichiometry between MICU1 and MCU could account for the previously described differences between heart and liver in mitochondrial Ca2+ uptake. Immunoblotting showed higher expression for all MICU1, MICU2 and MCU in mouse liver versus heart mitochondria, and a 4.5 fold higher MICU1 to MCU ratio in liver. In fluorometric measurements of mitochondrial Ca2+ uptake, heart mitochondria displayed a decreased threshold and lesser cooperativity compared to liver mitochondria. Additionally, NAD(P)H elevation was detectable after exposure to moderate [Ca2+] elevations only in heart mitochondria. Overexpression of MICU1 in the heart using AAV9-MICU1 tail-vein injection significantly increased the MICU1 protein level without any changes of MICU2 or MCU. This increased the MICU1 to MCU ratio in the heart and led to increased thresholding and cooperativity, reproducing the liver-like mitochondrial Ca2+ uptake phenotype. Vice versa MICU1 downregulation in the liver has been shown to lower the threshold and cooperativity of mitochondrial Ca2+ uptake in hepatocytes. Thus, heart and liver mitochondria show different levels of Ca2+ threshold and cooperative activation of Ca2+ uptake, which seem to result from differential quantitative relationship between MICU1 and MCU.

The first two authors equally contributed.Voltage Dependent Anion Channel (VDAC) is a pore forming protein located in outer mitochondrial membrane (1). Its lack is lethal in human (2) and it is involved in cellular cross-talk, important in the apoptotic cascade (3). Its structure is a transmembrane β-barrel (4) organized in 19 β-strands and a N-terminal α-helix probably important for gating (5). However the sidedness of VDAC inside the membrane still remains unresolved (4). This issue is essential in order to define the pore structural determinants interacting with soluble proteins. We expressed, in HeLa cells, a recombinant hVDAC1 carrying C-terminal tag including the hemagglutinin-tag (HA), the specific caspase 3/7 cleavage site (DEVD) and 7 histidine residues (7xHis). DEVD amino acidic sequence can be cleaved upon apoptosis induction by staurosporine. A complementary Fluorescence Protease Protection assay was performed where cell protein are exposed to protease digestion after plasma membrane permeabilization. If the C-terminus of VDAC1 is exposed to the cytosolic side, we expect to lose the His tag upon DEVD cleavage. As result only the HA can be detected at mitochondrial level. Conversely if the C-terminus is oriented towards the IMS, the DEVD is not cleavable and both tags will be detected in mitochondria. Our results by confocal microscopy and appropriate controls with other membrane-oriented constructs showed that this strategy is able to define the sidedness of the VDAC pore.1) Shoshan-Barmatz, V. et al (2010) Mol Aspects Med 31, 227-285.2) Huizing, M. et al (1994) 8924, 762-762.3) Tomasello M.F. et al (2009) Cell Res. 19, 1363-1376.4) De Pinto V. et al (2008) J. Bioenerg Biomemb 40, 139-147.5) De Pinto V. et al (2007) Chembiochem 8, 744-756.Acknowledgements to FIRB_PRIN Grants.

With the actual increasing menace of bacterial resistance, the antibiotic permeation mechanisms have been the centre of attention, in many research fields. OmpF is a pore forming protein, found in Gram-negative bacteria's outer membrane, known to have an important role in the uptake of nutrients and antibiotics (e.g. quinolones), towards the interior of the bacterial cell. Although several studies have been published of protein-drug interaction in the complex microbiological environment, an understanding of this mechanism at a molecular level is still lacking. Quinolones are one of the most prescribed classes of antibiotics, both in human and veterinary medicine, and this wide use seems to be the main cause for bacterial resistance. Fluoroquinolones were developed to increase quinolone antibacterial activity by changes in the quinolone structure. Enrofloxacin is a second generation fluoroquinolone being the first one to be introduced in Veterinary medicine. Its human analogue is the well known Ciprofloxacin. The main purpose of this work was to study Enrofloxacin permeation pathways. Permeation or interaction of antibiotics with lipid bilayers (liposomes) is of great importance in order to clarify the lipid component function's in membrane permeation. A first approach was the determination of partition coefficients Enrofloxacin/liposomes, which allowed the quantification of this interaction. After, OmpF was reconstituted in liposomes (proteoliposomes) by two different reconstitution methods (Gel exclusion chromatography and using detergent adsorbing beads) in order to compare size, homogeneity and protein orientation in proteoliposomes. Proteoliposomes quenching studies, using water soluble quencher KI and acrylamide, as well as ion conductance measurements were performed to establish the site of interaction/translocation of Enrofloxacin.

Calcium flux through store operated calcium entry (SOCE) is a major regulator of intracellular calcium homeostasis and various calcium signaling pathways. The two key components of the store operated calcium release activated calcium (CRAC) channel are the Ca2+ sensing protein stromal interaction molecule 1 (STIM1) and the channel pore forming protein Orai1. Following calcium depletion from the endoplasmic reticulum, STIM1 undergoes a series of conformational changes which unmask a minimal Orai1 activating domain called CAD. CAD binds to two sites in Orai1, one in the N’ terminus and one in the C’ terminus. The energetic coupling of binding of the STIM1 ligand to opening of the channel pore is poorly understood. In this study we show that both N’ and C’ terminal domains of Orai1 synergistically contribute to interaction with CAD to allosterically couple STIM1 binding with channel gating and modulation of ion selectivity.