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Mitochondrial free [Ca2+] increases during ATP/ADP antiport and ADP phosphorylation: exploration of mechanisms.

ABSTRACT: ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+](m)) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+](m) is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+](m), membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and P(i). Energized mitochondria showed a dose-dependent increase in [Ca2+](m) after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] ([Ca2+](e)); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased [Ca2+](m) up to twofold. The ADP effect on increasing [Ca2+](m) could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or P(i). Oligomycin largely reduced the increase in [Ca2+](m) by ADP compared to control, and [Ca2+](m) did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca2+](m) increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca2+](e). These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca2+](m) largely due to the interaction of matrix Ca2+ with ATP, ADP, P(i), and cation buffering proteins in the matrix.

SUBMITTER: Haumann J

PROVIDER: S-EPMC2920628 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Mitochondrial free [Ca2+] increases during ATP/ADP antiport and ADP phosphorylation: exploration of mechanisms.

Haumann Johan J Dash Ranjan K RK Stowe David F DF Boelens Age D AD Beard Daniel A DA Camara Amadou K S AK

Biophysical journal 20100801 4

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+](m)) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+](m) is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+](m), membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, ...[more]

PMID: 20712982

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Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

Dataset Information

Mitochondrial free [Ca2+] increases during ATP/ADP antiport and ADP phosphorylation: exploration of mechanisms.

Publications

Mitochondrial free [Ca2+] increases during ATP/ADP antiport and ADP phosphorylation: exploration of mechanisms.

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