Tissue-specific Subunits Research Articles

Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting.

The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents.

MICU1 Confers Protection from MCU-Dependent Manganese Toxicity

The mitochondrial calcium uniporter is a highly selective ion channel composed of species- and tissue-specific subunits. However, the functional role of each component still remains unclear. Here, we establish a synthetic biology approach to dissect the interdependence between the pore-forming subunit MCU and the calcium-sensing regulator MICU1. Correlated evolutionary patterns across 247 eukaryotes indicate that their co-occurrence may have conferred a positive fitness advantage. We find that, while the heterologous reconstitution of MCU and EMRE invivo in yeast enhances manganese stress, this is prevented by co-expression of MICU1. Accordingly, MICU1 deletion sensitizes human cells to manganese-dependent cell death by disinhibiting MCU-mediated manganese uptake. As a result, manganese overload increases oxidative stress, which can be effectively prevented by NAC treatment. Our study identifies a critical contribution of MICU1 to the uniporter selectivity, with important implications for patients with MICU1 deficiency, as well as neurological disorders arising upon chronic manganese exposure.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

Overexpression of protein phosphatase 2A in a murine model of chronic myocardial infarction leads to increased adverse remodeling but restores the regulation of β-catenin by glycogen synthase kinase 3β

Background/ObjectivesIncreased activity of cardiac protein phosphatases is an important feature in human heart failure. Several different protein phosphatases (PP) are involved in the regulation of excitation–contraction-coupling of the myocardium. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase consisting of a dimeric core enzyme and tissue-specific subunits. In this study we used transgenic mice overexpressing PP2A to further investigate the role of PP2A in cardiac remodeling after myocardial infarction. Methods and resultsAdult male CD-1 mice overexpressing the catalytic subunit α of PP2A (αMHC-PP2A; TG) underwent chronic LAD-ligation or sham surgery, respectively; wildtype littermates (WT) were used as controls. Cardiac function was determined by echocardiography before and 28days after LAD-ligation. 28days after MI, the animals were sacrificed and cardiac remodeling was analyzed in histological sections and by Western blots. PP2A overexpression leads to dilated cardiomyopathy in mice, and increased cardiomyocyte hypertrophy and fibrosis of the remote myocardium can be seen after myocardial infarction. However, we found an improved survival of TG in the subacute phase after MI in comparison to WT. On the molecular level, TG shows reduced expression of SERCA and CaMKII alpha both under basal condition as well 28days after MI. Additionally, the regulation of the Akt/GSK3β/β-catenin pathway is severely disturbed in TG at baseline where a significant activation of Akt is found that coincides with the typical phosphorylation of GSK3β. However, this does not lead to the accumulation of β-catenin — on the contrary: phosphorylation-induced degradation of β-catenin is significantly enhanced. ConclusionTransgenic overexpression of myocardial PP2A causes adverse remodeling which coincides with a disruption of the classical Akt/GSK3/β-catenin pathway under baseline conditions that is restored to normal values in chronic myocardial infarction. Even so overall survival of TG after myocardial infarction was not constrained and survival after day 2 post MI was improved.

“Cardiac K ATP ”

Understanding the etiology of and development of appropriate treatment for myocardial ischemia and associated sudden cardiac death is a major goal of cardiovascular research. In a recent review,1 a compelling case is presented that sarcolemmal ATP-sensitive potassium (KATP) channels are an excellent target for antiarrhythmic therapy. Because opening of cardiac KATP channels shortens the action potential and reduces the refractory period, its activation can establish an arrhythmogenic substrate, supporting reentry. Hence, inhibition of KATP could be an ideal way to stop or even prevent arrhythmias: these channels tend to be closed under normal circumstances and to open only when cell metabolism is inhibited; therefore, any agents that inhibit KATP activity should specifically target channels only during ischemia, leaving nonischemic myocardium unaffected. On the other hand, activation of cardiac KATP channels has consistently been shown to protect the heart from damage during ischemia by limiting Ca entry.2 Finding a way to balance these opposing effects on the heart before, during, and after an ischemic event remains an intractable issue. Until recently, efforts to manipulate cardiac KATP have generally been conducted under a single underlying assumption, namely that cardiac KATP channels are structurally and functionally the same throughout the heart. However, recent studies, including the new report of Bao and colleagues3 in this issue, reveal that “cardiac KATP” is not a single entity and is more accurately a collection of structurally distinct channels. The relative density of each one varies regionally in the heart, may differ between species, and may be under dynamic control. Article see p 926 KATP channels are expressed in many different excitable tissues, where they couple cell energetic status with membrane excitability and function.4 In different tissues, KATP channels exhibit different properties. This …

Expression of ATP-Sensitive Potassium (K ATP) Channel Subunits in Mammalian Liver

ATP-sensitive potassium (KATP) channels are expressed in multiple tissues, where they provide a link between cellular metabolism and membrane excitability. They consist of two subunits: the inward rectifier potassium channel Kir6, which forms the pore, and the sulfonylurea receptor SUR, an ATP-Binding Cassette (ABC) transporter that functions as the regulatory subunit. Differential expression of Kir6 and SUR isotypes leads to formation of KATP channels with distinct electrophysiological and pharmacological properties, and a number of tissue-specific subunit combinations have been described. Although KATP channels in the liver remain largely unexplored, there is some evidence that KATP channels are expressed in hepatocytes. However, a rigorous characterization of the subunit composition of hepatocytic KATP channels is still lacking. In order to address this, we performed end-point RT-PCR for KATP channel subunit mRNA in extracts from mouse and rat liver, as well as from the human-derived hepatocyte cell line HepG2. Interestingly, a range of expression patterns was observed. In mouse liver, Kir6.1 and SUR2 were detected, while no signal was seen for Kir6.2 and SUR1 (n=3-5). In rat liver, signal was detected for all major subunits: Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B (n=3). In HepG2 cells, signal was detected for Kir6.1, Kir6.2, and SUR2B, but not for SUR1 or SUR2A (n=3). While detection of Kir6.1 and SUR2/SUR2B in mouse and rat liver may reflect vascular smooth muscle channels, the presence of these subunits in HepG2 cells indicates that they may in fact represent KATP channels expressed in hepatocytes. Additionally, expression of Kir6.2 in both rat liver and HepG2 cells is suggestive of a Kir6.2-containing hepatocytic KATP channel. Ultimately, correlation with electrophysiological and pharmacological experiments will be necessary to elucidate the subunit composition of hepatocytic KATP channels, as well as their physiological role.

Cellular physiology of the renal H+ATPase

Vacuolar-type H+ATPases are multisubunit macromolecules that play an essential role in renal acid-base homeostasis. Other cellular processes also rely on the proton pumping ability of H+ATPases to acidify organellar or lumenal spaces. Several diseases, including distal renal tubular acidosis, osteoporosis and wrinkly skin syndrome, are due to mutations in genes encoding alternate subunits that make up the H+ATPase. This review highlights recent key articles in this research area. Further insights into the structure, expression and regulation of H+ATPases have been elucidated, within the kidney and elsewhere. This knowledge may enhance the potential for future drug targeting. Novel findings concerning tissue-specific subunits of the H+ATPase that are important in the kidney and more general lessons of H+ATPase function and regulation are slowly emerging, though the paucity of cellular tools available has to date limited progress.

V1 and V0 Domains of the Human H+-ATPase Are Linked by an Interaction between the G and a Subunits

The specialized H(+)-ATPases found in the inner ear and acid-handling cells in the renal collecting duct differ from those at other sites, as they contain tissue-specific subunits, such as a4 and B1, and in the kidney, C2, d2, and G3 as well. These subunits replace the ubiquitously expressed forms. Previously, we have shown that, in major organs of both mouse and man, G3 subunit expression is limited to the kidney. Here we have shown wide-spread transcription of murine G3 in specific segments of microdissected nephron, and demonstrated additional G3 expression in epithelial fragments from human inner ear. We raised a polyclonal G3-specific antibody, which specifically detects G3 from human, mouse, and rat kidney lysates, and displays no cross-reactivity with G1 or G2. However, immunolocalization using this antibody on human and mouse kidney sections was unachievable, suggesting epitope masking. Phage display analysis and subsequent enzyme-linked immunosorbent assay, using the G3 antibody epitope peptide as bait, identified a possible interaction between the G3 subunit and the a4 subunit of the H(+)-ATPase. This interaction was verified by successfully using purified, immobilized full-length G3 to pull down the a4 subunit from human kidney membrane preparations. This confirms that a4 and G3 are component subunits of the same proton pump and explains the observed epitope masking. This interaction was also found to be a more general feature of human H(+)-ATPases, as similar G1/a1, G3/a1, and G1/a4 interactions were also demonstrated. These interactions represent a novel link between the V(1) and V(0) domains in man, which is known to be required for H(+)-ATPase assembly and regulation.

Physiological and Metabolic Implications of V-ATPase Isoforms in the Kidney

Not all vacuolar-type H(+)-ATPases (V-ATPases) are alike; those responsible for H(+) movement across plasma membranes contain some different, tissue-specific subunit isoforms. This brief review outlines those that have special relevance to the kidney, and illustrates their importance by describing various human diseases where loss of local proton pump function not only confers a severe phenotype, but has revealed related tissues where these same isoforms are expressed, signifying their physiological importance.

A specific functional interaction between CHIF and Na,K-ATPase: role of FXYD proteins in the cellular regulation of the pump.

CHIF (corticosteroid hormone-induced factor) is a member of the FXYD family that shares approximately 50% homology with the gamma subunit of Na,K-ATPase. It is expressed in renal collecting duct and distal colon, and is upregulated by Na(+) deprivation and high K(+) diet. Both CHIF and gamma are coimmunoprecipitated by an anti-alpha subunit antibody, and alpha is immunoprecipitated by anti-gamma and anti-CHIF antibodies. (86)Rb(+) flux experiments in CHIF-transfected HeLa cells demonstrate that CHIF increases the affinity for cytoplasmic Na(+), but does not affect the affinity for extracellular K(Rb). A physiological role of CHIF in kidney function is further elucidated by the phenotypic analysis of CHIF knockout mice. Taken together with data by others, it appears that FXYD proteins are tissue-specific subunits or regulators of the Na,K-ATPase whose function is to adjust the pump kinetics to particular physiological needs.

Inherited disorders of the H+-ATPase.

The alpha-intercalated cell in the distal nephron shares a number of molecular features with the osteoclast, including site-limited proton pumps that are present at high density. These are multisubunit H -ATPases, which are essential for acid-base homeostasis and for the maintenance of normal bone turnover. In recent years it has become evident that some rare inherited human disorders are due to pump dysfunction in kidney or in bone; these are reviewed here. The present review provides an overview of acid secretion in both kidney and bone, and describes the recently identified diseases that are associated with mutations in tissue-specific subunits of these pumps. Elucidation of the molecular bases of a number of inherited renal acidopathies and bone disorders raises the possibility that additional tissue-specific subunits of these important pumps will be identified, gives hope for a better understanding of normal function at the molecular level, and may have implications for future therapeutic development.

Purification and characterisation of glutathione S-transferases from the freshwater clam Corbicula fluminea (Müller)

Glutathione S-transferases (GSTs) are involved in the phase II detoxification metabolism. To provide a molecular basis for their use as biomarkers of pollution, cytosolic GSTs from the freshwater clam Corbicula fluminea have been purified by glutathione-Sepharose affinity chromatography, anion-exchange chromatography (AEC) and reversed-phase (RP) HPLC. SDS-PAGE of visceral mass (VM) affinity-purified extracts revealed four subunits with apparent molecular masses (MW) of 30.2, 29.2, 28.5 and 27.2 kDa. Analysis by non-denaturing PAGE revealed three acidic dimeric proteins with apparent MW of 64, 55 and 45 kDa, named GSTc1, GSTc2 and GSTc3, respectively, based on their elution order by AEC. Only GSTc2 and GSTc3 exhibited GST activity towards 1-chloro-2,4-dinitrobenzene. A tissue-specific subunit pattern was obtained by RP-HPLC of affinity-purified extracts from VM and gills (GI): three major peaks were resolved, one of which was common to both tissues. MW of each VM subunit was determined by electrospray ionisation-mass spectrometry: 23602±1 Da for the major subunit and 23289±1 Da for the minor ones. Immunoblot analysis revealed all subunits from both tissues were related to the Pi-class GSTs. In addition, minor VM subunits were slightly related to the Mu-class ones. The interest of such molecular studies in biomonitoring programs is discussed.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Analysis and Thiol-Group Determination of Isoforms of Bovine Cytochrome c Oxidase, a Hydrophobic Multisubunit Membrane Protein

Matrix-assisted laser desorption/ionization–mass spectra (MALDI–MS) are obtained from entire bovine heart (H) and liver (L) cytochrome c oxidase membrane protein complexes. Molecular masses of most of the subunits are in excellent agreement with the published sequences. Some corrections are necessary for the nuclear coded subunit IX, which is N-acetylated, and X, with a corrected C-terminal peptide sequence. The mass values of two of the three tissue-specific subunits (VIII-L and X-L) are not in agreement with the DNA-deduced sequences and have been corrected by protein sequencing. For the investigation of the cysteine status 7-diethyl-amino-3-(4′-maleimidylphenyl)-4-methylcoumarin proved to be an excellent site-specific reagent. MALDI–MS with the SH-reacted enzyme indicates disulfide bridges only in subunit VII and a distorted tetrahedral S coordination of the zinc in subunit VI.

Tissue-specific subunit of the mouse cytosolic chaperonin-containing TCP-1

We have cloned a novel Tcp-1-related mouse testis cDNA encoding a polypeptide of 531 amino acids which shares 81.2% identity with the ζ subunit of the mouse cytosolic chaperonin-containing TCP-1 (CCT). Immunoblot analysis of mouse testis CCT subunits separated by 2-dimensional gel electrophoresis indicates that this novel gene, Cctz-2, encodes a CCT subunit of M r 57 000 and pI 7.1. Cctz-2 mRNA is detected only in testis whereas the other Cctz gene, Cctz-1, is expressed in all tissues investigated. The CCTζ-2 subunit may have specific functions in the folding of testicular proteins and for interactions with testicular molecular chaperones.

Species-specific expression of cytochrome c oxidase isozymes

Cytochrome c oxidase was isolated from livers and hearts of sheep, dog and rabbit, and the polypeptide composition was analyzed by two different SDS-PAGE separation systems. The gels were blotted on PVDF-membranes and the N-terminal amino acid sequences of the tissue-specific subunits VIa, VIIa and VIII were determined in a protein sequencer. Except for subunit VIIa from rat, subunits VIa and VIIa from all investigated mammals are tissue-specific expressed in liver and heart. In contrast, subunit VIII is clearly different in liver and heart of bovine, dog and rat, but identical in liver and heart of human (liver-type), sheep, rabbit and also in rainbow trout (heart-type). The data suggest a strong species-specific variation of the regulatory properties of cytochrome c oxidase in different tissues.

Dependence of Globin Gene Expression in Mouse Erythroleukemia Cells on the NF-E2 Heterodimer

High-level, tissue-specific expression of the beta-globin genes requires the presence of an upstream locus control region (LCR). The overall enhancer activity of the beta-globin complex LCR (beta-LCR) is dependent on the integrity of the tandem NF-E2 sites of HS-2. The NF-E2 protein which binds these sites is a heterodimeric basic leucine zipper protein composed of a tissue-specific subunit, p45 NF-E2, and a smaller subunit, p18 NF-E2, that is widely expressed. In these studies, we sought to investigate the role of NF-E2 in globin expression. We show that expression of a dominant-negative mutant p18 greatly reduces the amount of functional NF-E2 complex in the cell. Reduced levels of both alpha- and beta-globin were associated with the lower levels of NF-E2 activity in this cell line. Globin expression was fully restored upon the introduction of a tethered p45-p18 heterodimer. We also examined CB3 cells, a mouse erythroleukemia (MEL) cell line that does not express endogenous p45 NF-E2, and demonstrated that the restoration of globin gene expression was dependent upon the levels of expressed tethered NF-E2 heterodimer. Results of DNase I hypersensitivity mapping and in vivo footprinting assays showed no detectable chromatin alterations in beta-LCR HS-2 due to loss of NF-E2. Finally, we examined the specificity of NF-E2 for globin gene expression in MEL cells. These experiments indicate a critical role for the amino-terminal domain of p45 NF-E2 and show that a related protein, LCRF1, is unable to restore globin gene expression in p45 NF-E2-deficient cells. From these results, we conclude that NF-E2 is specifically required for high level goblin gene expression in MEL cells.