protonophore
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Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.
Oxidative stress and ROS are important players in the pathogenesis of numerous diseases. In addition to directly altering proteins, ROS also affects lipids with negative intrinsic curvature such as phosphatidylethanolamine (PE), producing PE adducts and lysolipids. The formation of PE adducts potentiates the protonophoric activity of mitochondrial uncoupling proteins, but the molecular mechanism remains unclear. Here, we linked the ROS-mediated change in lipid shape to the mechanical properties of the membrane and the function of uncoupling protein 1 (UCP1) and adenine nucleotide translocase 1 (ANT1). We show that the increase in the protonophoric activity of both proteins occurs due to the decrease in bending modulus in lipid bilayers in the presence of lysophosphatidylcholines (OPC and MPC) and PE adducts. Moreover, MD simulations showed that modified PEs and lysolipids change the lateral pressure profile of the membrane in the same direction and by the similar amplitude, indicating that modified PEs act as lipids with positive intrinsic curvature. Both results indicate that oxidative stress decreases stored curvature elastic stress (SCES) in the lipid bilayer membrane. We demonstrated that UCP1 and ANT1 sense SCES and proposed a novel regulatory mechanism for the function of these proteins. The new findings should draw the attention of the scientific community to this important and unexplored area of redox biochemistry.
- Publikační typ
- časopisecké články MeSH
2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in "diet pills", despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP's uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP's protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.
- MeSH
- 2,4-dinitrofenol farmakologie MeSH
- jaterní mitochondrie metabolismus MeSH
- krysa rodu rattus MeSH
- lipidové dvojvrstvy metabolismus MeSH
- membránové potenciály účinky léků MeSH
- mitochondriální ADP/ATP-translokasy metabolismus MeSH
- mitochondriální odpřahující proteiny metabolismus MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Yeast cells exhibit a negative surface potential due to negative charges at the cell membrane surface. Consequently, local concentrations of cations at the periplasmic membrane surface may be significantly increased compared to their bulk environment. However, in cell suspensions only bulk concentrations of cations can be measured directly. Here we present a novel method enabling the assessment of local pH at the periplasmic membrane surface which can be directly related to the underlying cell surface potential. In this proof of concept study using Saccharomyces cerevisiae cells with episomally expressed pH reporter, pHluorin, intracellular acidification induced by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was measured using synchronously scanned fluorescence spectroscopy (SSF). The analysis of titration curves revealed that the pH at the periplasmic surface of S. cerevisiae cells was about two units lower than the pH of bulk medium. This pH difference was significantly decreased by increasing the ionic strength of the bulk medium. The cell surface potential was estimated to amount to -130 mV. Comparable results were obtained also with another protonophore, pentachlorophenol (PCP).
- MeSH
- fluorescenční spektrometrie metody MeSH
- karbonylkyanid-m-chlorfenylhydrazon MeSH
- koncentrace vodíkových iontů * MeSH
- membránové potenciály * MeSH
- metody MeSH
- periplazma chemie MeSH
- Saccharomyces cerevisiae chemie cytologie MeSH
- zelené fluorescenční proteiny MeSH
- Publikační typ
- časopisecké články MeSH
Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (DeltaPsi(m)) during GSIS are lacking. Using high resolution respirometry and parallel DeltaPsi(m) monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and DeltaPsi(m) during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, DeltaPsi(m) jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.
- MeSH
- adenosindifosfát metabolismus MeSH
- adenosintrifosfát metabolismus MeSH
- financování organizované MeSH
- glukosa farmakologie MeSH
- inzulinom sekrece MeSH
- krysa rodu rattus MeSH
- kyselina linolová farmakologie MeSH
- Langerhansovy ostrůvky sekrece účinky léků MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondrie metabolismus účinky léků MeSH
- nádorové buňky kultivované MeSH
- nádory slinivky břišní sekrece MeSH
- potkani Wistar MeSH
- spotřeba kyslíku účinky léků MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
