Biomedical subjects

John Orlowski

Publications and source records attributed to John Orlowski.

8 recordsLinked to original sources

PubMed2005

Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: role in apical retention and targeting.

Proximal tubular reabsorption of filtered sodium by the sodium/hydrogen exchanger isoform 3 (NHE3), located on the apical membrane, is fundamental to the maintenance of systemic volume and pH homeostasis. NHE3 is finely regulated by a variety of hormones and by changes in ionic composition and volume, likely requiring redistribution of the exchangers. We analyzed the subcellular distribution and dynamics of the exchangers by generating an epithelial line expressing NHE3 tagged with an exofacial epitope, which enabled us to monitor exchanger mobility and traffic in intact cells. Using determinations of fluorescence recovery after photobleaching in combination with dynamic measurements of subcellular distribution, we found that, in renal epithelial cells, NHE3 exists in four distinct subcompartments: a virtually immobile subpopulation that is retained on the apical membrane by interaction with the actin cytoskeleton in a manner that depends on the sustained activity of Rho GTPases; a mobile subpopulation on the apical membrane, which can be readily internalized; and two intracellular compartments that can be differentiated by their rate of exchange with the apical pool of NHE3. We provide evidence that detachment of the immobile fraction from its cytoskeletal anchorage leads to rapid internalization. These observations suggest that modulation of the mobile fraction of NHE3 on the apical membrane can alter the number of functional exchangers on the cell surface and, consequently, the rate of transepithelial ion transport. Regulation of the interaction of NHE3 with the actin cytoskeleton can therefore provide a new mode of regulation of sodium and hydrogen transport.

Amiloride↗

PubMed2005

Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7.

The mammalian (Na(+),K(+))/H(+) exchanger NHE7 resides chiefly in the trans-Golgi network (TGN) and post-Golgi vesicles where it is thought to contribute to organellar pH homeostasis. However, the mechanisms that underlie the targeting and regulation of NHE7 are unknown. To gain insight into these processes, yeast two-hybrid methodology was used to screen a human brain cDNA library for proteins that interact with the cytoplasmic C-terminus of NHE7. One binding partner we identified was SCAMP2, a member of the secretory carrier membrane protein (SCAMP) gene family. Direct association of these two proteins was further supported by co-immunolocalization and co-immunoprecipitation analyses using transfected cells, by their co-sedimentation in membrane fractions resolved on sucrose density gradients, and by in vitro protein binding assays. Other members of the SCAMP family, such as SCAMP1 and SCAMP5, also associated with NHE7. The majority of the NHE7-SCAMP complexes accumulated at the TGN, but a minor fraction also resided in recycling vesicles. Biochemical analyses indicated that the C-terminal cytoplasmic tail of NHE7 bound preferentially to a highly conserved cytoplasmic loop between the second and the third transmembrane segments (TM2-TM3 loop) of SCAMP2. A deletion mutant of SCAMP2 lacking this region (SCAMP2/Delta184-208) bound weakly to NHE7, but caused a significant fraction of NHE7 and wild-type SCAMP2 to redistribute to a pool of scattered recycling vesicles without noticeably affecting the location of other resident TGN (syntaxin 6) or Golgi cisternae (GM130) proteins. Conversely, a GFP-tagged TM2-TM3 construct of SCAMP2 interacted with NHE7, but also led to the redistribution of NHE7 to dispersed vesicular structures. We propose a model wherein SCAMPs participate in the shuttling of NHE7 between recycling vesicles and the TGN.

Animals↗

PubMed2005

beta-Arrestins bind and decrease cell-surface abundance of the Na+/H+ exchanger NHE5 isoform.

The neuronal Na(+)/H(+) exchanger NHE5 isoform not only resides in the plasma membrane but also accumulates in recycling vesicles by means of clathrin-mediated endocytosis. To further investigate the underlying molecular mechanisms, a human brain cDNA library was screened for proteins that interact with the cytoplasmic C-terminal region of NHE5 by using yeast two-hybrid methodology. One candidate cDNA identified by this procedure encoded beta-arrestin2, a specialized adaptor/scaffolding protein required for internalization and signaling of members of the G protein-coupled receptor superfamily. Direct interaction between the two proteins was demonstrated in vitro by GST fusion protein pull-down assays. Sequences within the N-terminal receptor activation-recognition domain and the C-terminal secondary receptor-binding domain of beta-arrestin2 conferred strong binding to the C terminus of NHE5. Full-length NHE5 and beta-arrestin2 also associated in intact cells, as revealed by their coimmunoprecipitation from extracts of transfected CHO cells. Moreover, ectopic expression of both proteins caused a redistribution of beta-arrestin2 from the cytoplasm to vesicles containing NHE5, and significantly decreased the abundance of the transporter at the cell surface. Comparable results were also obtained for the beta-arrestin1 isoform. These data reveal a broader role for arrestins in the trafficking of integral plasma membrane proteins than previously recognized.

Animals↗

PubMed2005

Use of phospho-specific antibodies to determine the phosphorylation of endogenous Na+/H+ exchanger NHE3 at PKA consensus sites.

Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na(+)/H(+) exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.

Animals↗

PubMed2004

Inhibition and redistribution of NHE3, the apical Na+/H+ exchanger, by Clostridium difficile toxin B.

NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.

Animals↗

PubMed2003

Diversity of the mammalian sodium/proton exchanger SLC9 gene family.

Sodium/proton antiporters or exchangers (NHE) are integral membrane proteins present in most, if not all, living organisms. In mammals, these transporters chiefly catalyze the electroneutral exchange of Na(+) and H(+) down their respective concentration gradients and are crucial for numerous physiological processes, ranging from the fine control of intracellular pH and cell volume to systemic electrolyte, acid-base and fluid volume homeostasis. NHE activity also facilitates the progression of other cellular events such as adhesion, migration, and proliferation. Thus far, eight distinct NHE genes (NHE1/SLC9A1-NHE8/SLC9A8) and several pseudogenes have been identified in the human genome. The functional genes encode proteins of varying primary sequence identity (25-70%), but share a common predicted secondary structure comprising 12 conserved membrane-spanning segments at the amino-terminus and a more divergent, cytoplasmically-oriented, carboxy-terminus. They show considerable heterogeneity in their patterns of tissue/cell expression and membrane localization. Functional studies have revealed further differences in their kinetic properties, sensitivity to pharmacological antagonists, and regulation by diverse hormonal and mechanical stimuli. Altered NHE activity has been linked to the pathogenesis of several diseases, including essential hypertension, congenital secretory diarrhea, diabetes, and tissue damage caused by ischemia/reperfusion. Further characterization of their functional properties should lead to a better understanding of their unique contributions to human health and disease.

Animals↗

PubMed2002

Clathrin-mediated endocytosis and recycling of the neuron-specific Na+/H+ exchanger NHE5 isoform. Regulation by phosphatidylinositol 3'-kinase and the actin cytoskeleton.

Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3'-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly.

Actins↗

PubMed2002

A slow pH-dependent conformational transition underlies a novel mode of activation of the epithelial Na+/H+ exchanger-3 isoform.

Allosteric control of Na(+)/H(+) exchange by intracellular protons ensures rapid and accurate regulation of the intracellular pH. Although this allosteric effect was heretofore thought to occur almost instantaneously, we report here the occurrence of a slower secondary activation of the epithelial Na(+)/H(+) exchanger (NHE)-3 isoform. This slow activation mode developed over the course of minutes and was unique to NHE3 and the closely related isoform NHE5, but was not observed in NHE1 or NHE2. Activation of NHE3 was not due to increased density of exchangers at the cell surface, nor was it accompanied by detectable changes in phosphorylation. The association of NHE3 with the cytoskeleton, assessed by its retention in the detergent-insoluble fraction, was similarly unaffected by acidification. In contrast to the slow progressive activation elicited by acidification, deactivation occurred very rapidly upon restoration of the physiological pH. We propose that NHE3 undergoes a slow pH-dependent transition from a less active to a more active state, likely by changing its conformation or state of association.

Allosteric Regulation↗