Mitochondria play a key role in cytosolic Ca2+ regulation and buffering, with Ca2+ uptake mainly via the inner membrane mitochondrial Ca2+ uniporter (MCU), and efflux of mitochondrial Ca2+ largely via the Na+/Ca2+ exchanger (mNCE). Previous reports have shown brain synaptic and non‐synaptic mitochondria display marked differences in Ca2+ uptake before permeabilization of the mitochondrial membranes, the so‐called permeable transition pore (mPTP). However, the kinetics of mitochondrial Ca2+ handling and its implications for Ca2+ buffering after boluses of CaCl2 have not been reported. In this study, we aimed to define the kinetics of Ca2+ handling in synaptic and non‐synaptic mitochondria isolated from rat brains via discontinuous percoll centrifugation followed by differential centrifugation. Respiratory control index (RCI), and subsequent changes in calcium retention capacity (CRC) and membrane potential (ΔΨm), were assessed in isolated mitochondria. CRC and ΔΨm were evaluated with Fura 4F penta‐K salt and TMRM dyes, respectively, using fluorescence spectrophotometry (Photon Technology). Mitochondria were energized with glutamate and malate, and CRC and ΔΨm were assessed during state 2 respiration. To investigate the potential role of the mNCE in Ca2+ handling, the NCE blocker CGP37157 (CGP) was used. In addition, experiments were conducted to determine the expression levels of the MCU and mNCE in the two mitochondria pools using western blot. Mitochondrial RCI was higher in the synaptic group compared to the non‐synaptic group, which is consistent with past results. Ca2+ uptake and retention without CGP were lower in synaptic mitochondria compared to non‐synaptic mitochondria. Addition of CGP markedly enhanced CRC in the synaptic mitochondria to levels found in non‐synaptic mitochondria without CGP. In contrast, the CGP induced changes in CRC were not observed in the non‐synaptic mitochondria. Membrane potential depolarization occurred in both synaptic and non‐synaptic mitochondria as matrix free Ca2+ accumulated, reflecting the decrease in Ca2+ sequestration or Ca2+ efflux in the non‐synaptic and synaptic mitochondria, respectively. MCU expression was higher in the non‐synaptic mitochondria compared to the synaptic mitochondria, whereas mNCE expression was not different. The increased Ca2+ uptake and retention in the presence of CGP in the synaptic mitochondria implies that mNCE is active in synaptic mitochondria, which leads to faster extrusion of added Ca2+ before it is sequestered. The lack of change in Ca2+ kinetics in non‐synaptic mitochondria suggests that NCE activity is lower. The ΔΨm and CRC results likely indicate that synaptic mitochondria can buffer Ca2+ like that in non‐synaptic mitochondria, but only do so when the mNCE is blocked. We propose that synaptic mitochondria may have increased mNCE activity to regulate its matrix free Ca2+, while non‐synaptic mitochondria depend mostly on buffering of the free Ca2+; the implications for these differential responses may be related to the physiological role of these two populations of mitochondria.Support or Funding InformationFunding: NIH T35 HL072483 and MCW‐AHWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alkylureas display hydrocarbon and amide groups, the primary functional groups of proteins. To obtain the thermodynamic information that is needed to analyze interactions of amides and proteins with nucleobases and nucleic acids, we quantify preferential interactions of alkylureas with nucleobases differing in the amount and composition of water-accessible surface area (ASA) by solubility assays. Using an established additive ASA-based analysis, we interpret these thermodynamic results to determine interactions of each alkylurea with five types of nucleobase unified atoms (carbonyl sp2O, amino sp3N, ring sp2N, methyl sp3C, and ring sp2C). All alkylureas interact favorably with nucleobase sp2C and sp3C atoms; these interactions become more favorable with an increasing level of alkylation of urea. Interactions with nucleobase sp2O are most favorable for urea, less favorable for methylurea and ethylurea, and unfavorable for dialkylated ureas. Contributions to overall alkylurea-nucleobase interactions from interactions with each nucleobase atom type are proportional to the ASA of that atom type with proportionality constant (interaction strength) α, as observed previously for urea. Trends in α-values for interactions of alkylureas with nucleobase atom types parallel those for corresponding amide compound atom types, offset because nucleobase α-values are more favorable. Comparisons between ethylated and methylated ureas show interactions of amide compound sp3C with nucleobase sp2C, sp3C, sp2N, and sp3N atoms are favorable while amide sp3C-nucleobase sp2O interactions are unfavorable. Strongly favorable interactions of urea with nucleobase sp2O but weakly favorable interactions with nucleobase sp3N indicate that amide sp2N-nucleobase sp2O and nucleobase sp3N-amide sp2O hydrogen bonding (NH···O═C) interactions are favorable while amide sp2N-nucleobase sp3N interactions are unfavorable. These favorable amide-nucleobase hydrogen bonding interactions are prevalent in specific protein-nucleotide complexes.

Quantitative information about amide interactions in water is needed to understand their contributions to protein folding and amide effects on aqueous processes and to compare with computer simulations. Here we quantify interactions of urea, alkylated ureas, and other amides by osmometry and amide-aromatic hydrocarbon interactions by solubility. Analysis of these data yields strengths of interaction of ureas and naphthalene with amide sp2O, amide sp2N, aliphatic sp3C, and amide and aromatic sp2C unified atoms in water. Interactions of amide sp2O with urea and naphthalene are favorable, while amide sp2O-alkylurea interactions are unfavorable, becoming more unfavorable with increasing alkylation. Hence, amide sp2O-amide sp2N interactions (proposed n-σ* hydrogen bond) and amide sp2O-aromatic sp2C (proposed n-π*) interactions are favorable in water, while amide sp2O-sp3C interactions are unfavorable. Interactions of all ureas with sp3C and amide sp2N are favorable and increase in strength with increasing alkylation, indicating favorable sp3C-amide sp2N and sp3C-sp3C interactions. Naphthalene results show that aromatic sp2C-amide sp2N interactions in water are unfavorable while sp2C-sp3C interactions are favorable. These results allow interactions of amide and hydrocarbon moieties and effects of urea and alkylureas on aqueous processes to be predicted or interpreted in terms of structural information. We predict strengths of favorable urea-benzene and N-methylacetamide interactions from experimental information to compare with simulations and indicate how amounts of hydrocarbon and amide surfaces buried in protein folding and other biopolymer processes and transition states can be determined from analysis of urea and diethylurea effects on equilibrium and rate constants.

Interactions between biochemical functional groups are fundamental to biopolymer self-assembly processes and enzyme-substrate binding and ligand binding, and a key determinant of biopolymer structure and function. To date little quantitative information has been available about their strengths and contributions to stability. Interactions of small solutes have been shown to be composed of additive contributions from interactions of one solute with individual function groups on the other solute (one way interactions). Here we test the hypothesis that solute-solute interactions in water can be dissected using additivity and either an ASA or weighted group-based analysis into contributions from group-group interactions (two way analysis). Data for preferential interactions (μ23 values) of amides with other amides and with aromatics and nucleobases as well as for interactions of alcohols and polyols with aromatics and nucleobases are obtained by osmometry and solubility assays and interpreted to obtain two way potentials (alpha values) for the group group interactions. Some of the most favorable interactions are those that correspond to hydrogen bonding between NH and C=O groups like those in protein 2o structures and in DNA/RNA duplexes. The interaction of aromatic C with amide O is very favorable, providing a second example of a favorable n-π∗ interaction, like that of amide C with amide O (Ref 1). Interactions of amides with Na+ and K+ salts of Hofmeister anions reveal that interactions of these salts with hydrocarbon C and amide N, follow the Hofmeister order (KSCN > KCl > KF; NaClO4 > NaCl > Na2SO4). These data quantify interaction of these salts with the most important protein groups and are useful to interpret or predict Hofmeister effects on protein unfolding m-values. 1) Bartlett GJ, Choudhary A, Raines RT, Woolfson DN (2010) Nat Chem Biol 6:615–620.