Extracellular acidification induces ROS- and mPTP-mediated death in HEK293 cells.

The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.

Digitalis-like Compounds Facilitate Non-Medullary Thyroid Cancer Redifferentiation through Intracellular Ca2+, FOS, and Autophagy-Dependent Pathways.

Up to 20%-30% of patients with metastatic non-medullary thyroid cancer have persistent or recurrent disease resulting from tumor dedifferentiation. Tumor redifferentiation to restore sensitivity to radioactive iodide (RAI) therapy is considered a promising strategy to overcome RAI resistance. Autophagy has emerged as an important mechanism in cancer dedifferentiation.

ATPase Activity Measurements Using Radiolabeled ATP.

ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [γ-(32)P]ATP, which is hydrolyzed by P-type ATPases to ADP and (32)Pi.

Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System.

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection.

Complex I and complex III inhibition specifically increase cytosolic hydrogen peroxide levels without inducing oxidative stress in HEK293 cells.

Inhibitor studies with isolated mitochondria demonstrated that complex I (CI) and III (CIII) of the electron transport chain (ETC) can act as relevant sources of mitochondrial reactive oxygen species (ROS). Here we studied ROS generation and oxidative stress induction during chronic (24h) inhibition of CI and CIII using rotenone (ROT) and antimycin A (AA), respectively, in intact HEK293 cells. Both inhibitors stimulated oxidation of the ROS sensor hydroethidine (HEt) and increased mitochondrial NAD(P)H levels without major effects on cell viability.

Na(+),K(+)-ATPase isoform selectivity for digitalis-like compounds is determined by two amino acids in the first extracellular loop.

Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na(+),K(+)-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin.

A missense variant of the ATP1A2 gene is associated with a novel phenotype of progressive sensorineural hearing loss associated with migraine.

Hereditary sensorineural hearing loss is an extremely clinical and genetic heterogeneous disorder in humans. Especially, syndromic hearing loss is subdivided by combinations of various phenotypes, and each subtype is related to different genes. We present a new form of progressive hearing loss with migraine found to be associated with a variant in the ATP1A2 gene.

Biochemical characterization of sporadic/familial hemiplegic migraine mutations.

Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na(+),K(+)-ATPase α2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed.

Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding.

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system.

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals.

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects.

Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions.

Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the α2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally.

Subunit-specific incorporation efficiency and kinetics in mitochondrial complex I homeostasis.

Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse.

Trolox-sensitive reactive oxygen species regulate mitochondrial morphology, oxidative phosphorylation and cytosolic calcium handling in healthy cells.

Aims: Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic calcium (Ca(2+)) handling in healthy human skin fibroblasts.

Results And Innovation: Trolox treatment reduced the levels of 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein (CM-H(2)DCF) oxidizing ROS, lowered cellular lipid peroxidation, and induced a less oxidized mitochondrial thiol redox state.

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts.

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice.

Na,K-ATPase activity modulates Src activation: a role for ATP/ADP ratio.

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src.

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits.

Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly.

Reprint of: Effective high-throughput overproduction of membrane proteins in Escherichia coli.

Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli.

Quantitative glucose and ATP sensing in mammalian cells.

The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic dysfunction plays an important role in many human diseases, including diabetes, cancer, inherited mitochondrial disorders, and metabolic syndrome. The monosaccharide glucose constitutes a key source of cellular energy.

Solute diffusion is hindered in the mitochondrial matrix.

Intracellular chemical reactions generally constitute reaction-diffusion systems located inside nanostructured compartments like the cytosol, nucleus, endoplasmic reticulum, Golgi, and mitochondrion. Understanding the properties of such systems requires quantitative information about solute diffusion. Here we present a novel approach that allows determination of the solvent-dependent solute diffusion constant (D(solvent)) inside cell compartments with an experimentally quantifiable nanostructure.

FXYD2 and Na,K-ATPase expression in isolated human proximal tubular cells: disturbed upregulation on renal hypomagnesemia?

Autosomal dominant renal hypomagnesemia (OMIM 154020), associated with hypocalciuria, has been linked to a 121G to A mutation in the FXYD2 gene. To gain insight into the molecular mechanisms linking this mutation to the clinical phenotype, we studied isolated proximal tubular cells from urine of a patient and a healthy subject. Cells were immortalized and used to assess the effects of hypertonicity-induced overexpression of FXYD2 on amount, activity and apparent affinities for Na(+), K(+) and ATP of Na,K-ATPase.

Chapter 16 The use of fluorescence correlation spectroscopy to probe mitochondrial mobility and intramatrix protein diffusion.

Within cells, functional changes in mitochondrial metabolic state are associated with alterations in organelle mobility, shape, and configuration of the mitochondrial matrix. Fluorescence correlation spectroscopy (FCS) is a technique that measures intensity fluctuations caused by single fluorescent molecules moving through a small detection volume. By mathematically correlating these fluctuations, information can be obtained about the concentration and rate of diffusion of the fluorescent molecules.

Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells.

Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved.

Effective high-throughput overproduction of membrane proteins in Escherichia coli.

Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli.

NDUFA2 complex I mutation leads to Leigh disease.

Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5.

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase.

Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.