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Larry Fliegel

Publications and source records attributed to Larry Fliegel.

29 records · Page 2Linked to original sources
PubMed2003

The Na+/H+ exchanger cytoplasmic tail: structure, function, and interactions with tescalcin.

We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.

Amino Acid Sequence↗
PubMed2003

Transcriptional regulation of Na+/H+ exchanger expression in the intact mouse.

We examined regulation of the Na+/H+ exchanger (NHE1 isoform) in the developing mouse. We generated transgenic mice with the Na+/H+ exchanger promoter directing expression of the beta-Galactosidase reporter. We found that expression of the Na+/H+ exchanger was maximum in the heart and liver of 12-day-old embryonic mice. Similar results were found in mice using the green fluorescent protein reporter driven by the Na+/H+ exchanger promoter. Detailed examination of the myocardium revealed that the GFP reporter protein was expressed in the cytoplasm of cardiomyocyte cells. We examined NHE1 protein expression in transgenic mice lacking the transcription factors AP-2alpha or the transcription factor COUP-TF1. Eighteen-day-old AP-2alpha heterozygote mice show no large changes in NHE1 expression in heart, lung, liver, kidney and brain. In contrast, 18-day-old embryos from AP-2alpha null mice showed a large increase in Na+/H+ exchanger protein expression in the brain. NHE1 protein levels in COUP-TF1 knockout embryos did not differ from wild type embryos. The results suggest that AP-2alpha and COUP-TF1 are not critical to NHE1 expression in the late stage embryo and that other related transcription factors may function in regulation of the Na+/H+ exchanger.

Animals↗
PubMed2003

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation.

The Na+/H+ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na+/H+ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na+/H+ exchanger were examined for their ability to translocate protons as assessed by a Cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na+/H+ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na+/H+ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na+/H+ exchangers.

Amino Acids↗
PubMed2002

Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger.

We examined the ability of carbonic anhydrase II to bind to and affect the transport efficiency of the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. The C-terminal region of NHE1 was expressed in Escherichia coli fused with an N-terminal glutathionine S-transferase or with a C-terminal polyhistidine tag. Using a microtiter plate binding assay we showed that the C-terminal region of NHE1 binds carbonic anhydrase II (CAII) and binding was stimulated by low pH and blocked by antibodies against the C-terminal of NHE1. The binding to NHE1 was confirmed by demonstrating protein-protein interaction using affinity blotting with CAII and immobilized NHE1 fusion proteins. CAII co-immunoprecipitated with NHE1 from CHO cells suggesting the proteins form a complex in vivo. In cells expressing CAII and NHE1, the H(+) transport rate was almost 2-fold greater than in cells expressing NHE1 alone. The CAII inhibitor acetazolamide significantly decreased the H(+) transport rate of NHE1 and transfection with a dominant negative CAII inhibited NHE1 activity. Phosphorylation of the C-terminal of NHE1 greatly increased the binding of CAII. Our study suggests that NHE1 transport efficiency is influenced by CAII, likely through a direct interaction at the C-terminal region. Regulation of NHE1 activity by phosphorylation could involve modulation of CAII binding.

Animals↗
PubMed2002

Thyroid hormone receptor alpha 1 regulates expression of the Na+/H+ exchanger (NHE1).

In this paper we examine the role of thyroid hormone in regulating expression of the Na+/H+ exchanger. Thyroid hormone has been reported to regulate the activity of the Na+/H+ exchanger messenger RNA in some cell types. Treatment of cardiac myocytes with 3,5',3'-triiodothyronine results in an increased expression of Na+/H+ exchanger protein. Also, compared with euthyroid animals, hypothyroid rats express decreased amounts of the Na+/H+ exchanger protein. To examine the mechanisms involved in regulating expression of the Na+/H+ exchanger, we have characterized the regulation of a distal element of the NHE1 promoter by the thyroid hormone receptor. We have previously shown that a -1085/-800 nucleotide (nt) region of the promoter is a modular element with a -841/-800 nt activating element. Using electrophoretic mobility shift assay, we show that this element interacts with thyroid hormone receptor TRalpha(1), a nuclear hormone receptor. The addition of exogenous TRalpha increased transcriptional activity of the -841/-800 nt element of the Na+/H+ exchanger promoter. We show that TRalpha binds to a region on the -841/-800 nt element that is near, but not identical, to the previously identified chicken ovalbumin upstream promoter transcription factor-binding site. Our results are the first demonstration that thyroid hormone and the thyroid hormone receptor TRalpha(1) regulate expression of the Na+/H+ exchanger.

3T3 Cells↗
PubMed2002

Properties of the Na+/H+ exchanger protein. Detergent-resistant aggregation and membrane microdistribution.

The Na+/H+ exchanger is a ubiquitous membrane protein of bacteria, plants and mammals. The first isoform discovered (NHE1) is present on the mammalian plasma membrane and transports one H+ out of cells in exchange for one extracellular Na+. With solubilization in standard SDS/PAGE buffer, this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDS or 2-mercaptoethanol. We examined the distribution of the Na+/H+ exchanger within membrane subfractions. The Na+/H+ exchanger was found both in caveolin-containing fractions and, in lesser amounts, in higher density membrane fractions where the bulk of proteins were contained. Treatment with cytochalasin D caused only a minor reduction of the amount of Na+/H+ exchanger present in caveolin-enriched fractions suggesting an intact cytoskeleton was not important for NHE1 localization to these microdomains. Treatment of cells with methyl beta-cyclodextrin had a small stimulatory effect on Na+/H+ exchanger activity and reduced the amount of Na+/H+ exchanger in low density membrane fractions. Our study demonstrates that SDS cannot maintain the protein in a monomeric state suggesting that strong hydrophobic interactions are responsible for this temperature dependent aggregation behavior. In addition a large proportion of the Na+/H+ exchanger protein is found to be enriched in low density caveolin-containing fractions.

Animals↗
PubMed2002

Structure and function of the NHE1 isoform of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cytoskeletal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.

Animals↗
PubMed2002

Developmental regulation of Na(+)/H(+) exchanger expression in fetal and neonatal mice.

We examined the hypothesis that Na(+)/H(+) exchanger expression is regulated during fetal and neonatal development and differentiation. To examine transcriptional regulation of the NHE1 isoform of the Na(+)/H(+) exchanger, transgenic mice were created that contained the mouse NHE1 promoter driving expression of green fluorescent protein. The level of NHE1 transcription varied between tissues and with the stage of embryonic development. The highest expression was in the heart and liver of 12- to 15-day-old mice, and this declined with age. To examine Na(+)/H(+) exchanger protein levels, we immunoblotted mouse tissues from 18-day-old embryos, neonates, and adults. Protein levels increased after embryonic day 18 and peaked at 14 days of age in the heart, lung, liver, kidney, and brain. The greatest rise in NHE1 protein expression occurred in the heart, whereas the smallest increase was in the brain. The results suggest that Na(+)/H(+) exchanger transcription and protein levels are controlled in a tissue-specific and time-dependent manner during development.

Aging↗
PubMed2002

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway.

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.

Animals↗
PubMedDate not supplied

Nanoliter sample handling combined with microspot MALDI-MS for detection of gel-separated phosphoproteins.

We describe a microspot matrix-assisted laser desorption ionization (MALDI) mass spectrometric approach to analyze gel-separated phosphoproteins. This method involves in-gel digestion of phosphoproteins after gel separation, followed by open tubular capillary (OTC) immobilized metal-ion affinity chromatography (IMAC) to capture the phosphopeptides with markedly reduced interferences from nonphosphorylated peptides. Nanoliter-volume of ammonium phosphate is used to elute the phosphopeptides captured on the capillary tube. After mixing with a small volume of matrix solution in the capillary, the effluent is deposited in a microspot on a sample plate for MALDI-MS analysis. It is also shown that, with peptide esterification after in-gel digestion of a phosphoprotein, negative ion detection in MALDI gives a distinct advantage over the positive ion mode of operation for phosphopeptide analysis, even without IMAC enrichment. However, the OTC-IMAC technique is demonstrated to be superior to the approach of negative ion detection of esterified in-gel digests without IMAC. OTC-IMAC is found to be sufficiently selective to capture phosphopeptides from in-gel digest of a gel band containing predominately one protein and the combination of peptide esterification and IMAC enrichment does not provide any real advantage. Using a standard phosphoprotein alpha-casein as a model system, we demonstrate that this OTC-IMAC method can detect a number of phosphopeptides after in-gel digestion with mid-fmol protein sample loading. An example of real world applications of this method is illustrated in the characterization of a fusion protein, His182, expressed in E. coli.

Amino Acid Sequence↗