Biomedical subjects

J Noël

Publications and source records attributed to J Noël.

At least 19 recordsLinked to original sources

PubMed2003

Anterior displacement correlates with neurological impairment in cervical facet dislocations.

We studied all patients with either unifacetal or bifacetal dislocations treated in our National Spinal Injuries Unit between January 1996 and December 2000. There were 25 cases, of which 13 were unifacetal and 12 bifacetal. Craniocervical traction was employed in 21 cases and closed reduction achieved in 11. Eighteen patients underwent surgical stabilisation. Traction weights of up to 36 kg were employed, but there was no relationship found between the level of dislocation and traction weight. Anterior translation was measured by a newly described method, and we found a statistically significant correlation between the neurological score on admission and the degree of anterior translation.

Adolescent↗

PubMed2002

Lisfranc injuries: patient- and physician-based functional outcomes.

The purpose of this study was to assess functional outcome of patients with a Lisfranc fracture dislocation of the foot by applying validated patient- and physician-based scoring systems and to compare these outcome tools. Of 25 injuries sustained by 24 patients treated in our institution between January 1995 and June 2001, 16 were available for review with a mean follow-up period of 36 (10-74) months. Injuries were classified according to Myerson. Outcome instruments used were: (a) Medical Outcomes Study 36-Item Short Form Health Survey (SF-36), (b) Baltimore Painful Foot score (PFS) and (c) American Orthopedic Foot and Ankle Society (AOFAS) mid-foot scoring scale. Four patients had an excellent outcome on the PFS scale, seven were classified as good, three fair and two poor. There was a statistically significant correlation between the PFS and Role Physical (RP) element of the SF-36.

Adolescent↗

PubMed2001

Protein changes in post mortem sea bass (Dicentrarchus labrax) muscle monitored by one- and two-dimensional gel electrophoresis.

This study was devoted to the identification of specific peptides and proteins which can be used as indicators of freshness in fish. The post mortem evolution of protein patterns in farmed sea bass muscle was monitored by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) after 0, 2, 4, and 6 days cold storage. SDS-electrophoresis, of total proteins and proteins soluble in low-ionic-strength solutions, revealed the gradual disappearance of a protein band of 16 kDa immediately after fish death. 2-DE allowed the classification of fish samples according to post mortem time. Three spots of interest, which disappeared progressively, were identified on the 2-DE patterns. Further research is required to establish the identity of these polypeptides and to evaluate their expression and post mortem evolution in another fish species.

Animals↗

PubMed2001

beta-Adrenergic stimulation of rat cardiac fibroblasts promotes protein synthesis via the activation of phosphatidylinositol 3-kinase.

beta -adrenergic agonists stimulate neonatal rat cardiac fibroblast growth, albeit the identity of the signaling event(s) remains equivocal. Isoproterenol (ISO) treatment increased intracellular cyclic AMP levels; however, cyclic AMP-elevating agents had no effect on protein synthesis. The tyrosine kinase inhibitor tyrphostin A25, and the inhibition of ras processing by the farnesyltransferase inhibitor BMS-191563 attenuated ISO-stimulated protein synthesis. Concomitant with increased protein synthesis, ISO stimulated extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) activity. The MEK1/2 inhibitor PD098059 abrogated ISO-stimulated ERK activity, albeit the increase in protein synthesis was unaffected. By contrast, LY294002 inhibited both ISO-stimulated PI3-K activity, and protein synthesis. ISO treatment did not increase the expression of transforming growth factor-beta(1)(TGF-beta(1)) mRNA, whereas a significant decrease in the steady-state mRNA level of TGF- beta(3)was observed. This latter effect was mimicked by cyclic AMP-elevating agents. Angiotensin II (AII) activation of the AT(1)receptor increased protein synthesis, but in contrast to ISO, the growth response was not inhibited by either tyrphostin A25 or BMS-191563, and was associated with the concomitant expression of both TGF-beta(1)and TGF-beta(3)mRNAs. Analogous to ISO, AII treatment increased ERK and PI3-K activity, and PI3-K was required for protein synthesis. These findings are the first to highlight the activation of PI3-K by a Gs(alpha)-coupled receptor, and its essential role in beta -adrenergic as well as AT(1)receptor-mediated protein synthesis in neonatal rat cardiac fibroblasts. However, despite the conserved role of PI3-K, additional disparate signaling pathways are recruited by ISO and AII, which may differentially influence fibroblast phenotype.

Adrenergic beta-Agonists↗

PubMed2001

Transient synaptic activation of NMDA receptors leads to the insertion of native AMPA receptors at hippocampal neuronal plasma membranes.

The molecular mechanisms underlying long-term potentiation (LTP) of excitatory synaptic transmission in the hippocampus are not well understood. Transient depolarisation of cultured postnatal hippocampal neurones (3x1 s exposure to 90 mM K+) induces a form of LTP that is manifest primarily as an increase in mEPSC frequency. Site-directed antibodies that recognise an extracellular region of all AMPA receptor (AMPAR) subunits (GluR1-4) were used for the immunolabelling of living neurones. These antibodies were raised in two species to enable sequential immunofluorescent labelling of individual living neurones before and after the induction of LTP. High K+ treatment resulted in the appearance of new AMPAR clusters at sites on the neuronal surface that previously lacked detectable AMPARs. The appearance of new AMPAR clusters was NMDA receptor (NMDAR)-dependent since it was antagonised by the application of NMDAR antagonists. Our data indicate that the transient synaptic activation of NMDARs can lead to the insertion of native AMPARs at sites on the neuronal membrane that initially lacks AMPARs.

Animals↗

PubMed2001

An electrophysiological characterisation of long-term potentiation in cultured dissociated hippocampal neurones.

Long-term potentiation (LTP) of synaptic transmission is under intense investigation. It is believed that the mechanisms involved in its induction and expression are critically involved in synaptic processes that are important for learning and memory and other physiological functions. A reliable means of inducing LTP in dissociated cultured neurones would facilitate investigations into the molecular basis of LTP but has been hard to achieve. Here we report a mechanism for inducing LTP in postnatal dissociated hippocampal neurones using transient depolarisation. This form of LTP is prevented by NMDA receptor antagonists and by chelating Ca2+ in the postsynaptic neurone. It is manifest primarily as an increase in the frequency of mEPSCs.

Animals↗

PubMed2000

Developmental changes in synaptic AMPA and NMDA receptor distribution and AMPA receptor subunit composition in living hippocampal neurons.

AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic increase was observed in the surface expression of AMPA receptors, but not NMDA receptors, between 3 and 10 d in culture. Immunocytochemical analysis of hippocampal neurons between 3 and 20 d in vitro shows no change in the proportion of synapses expressing NMDA receptors (approximately 60%) but a dramatic increase (approximately 50%) in the proportion of them that also express AMPA receptors. Furthermore, over this period the proportion of AMPA receptor-positive synapses expressing the GluR2 subunit increased from approximately 67 to approximately 96%. These changes will dramatically alter the functional properties of hippocampal synapses.

Animals↗

PubMed2000

Neutral calcium-activated proteases from European sea bass (Dicentrarchus labrax L.) muscle: polymorphism and biochemical studies.

Calcium-dependent proteinases or calpains were studied in fish muscle. Hydrophobic chromatography, followed by anion-exchange chromatography of the soluble fraction of sea bass white muscle proteins, resulted in three peaks of calcium-dependent protease activity at neutral pH (A, B and C). They are all neutral cysteine calcium-activated proteinases and can, therefore, be classified as calpain-like enzymes. From the Ca2+ concentration required for activity, A is a mu-calpain, and B and C are m-calpains. They share many properties with calpains from other vertebrate cells but differ in native mass, subunit composition, and the unusual numbers in which they are present. Their specific pattern of expression throughout the year could be of great importance to the resulting rate and extent of degradation of fish flesh after death.

Animals↗

PubMed2000

Regulation of the formation of tumor cell pseudopodia by the Na(+)/H(+) exchanger NHE1.

The Na(+)/H(+) exchanger NHE1 is involved in intracellular pH homeostasis and cell volume regulation and accumulates with actin in the lamellipodia of fibroblasts. In order to determine the role of NHE1 following epithelial transformation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) transformed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV). Expression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with b-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-mediated (22)Na uptake to ethylisopropylamiloride, a specific inhibitor of NHE1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intracellular pH induced upon EIPA treatment were also of higher magnitude in MSV-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 microM ethylisopropylamiloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells, the effect of ethylisopropylamiloride treatment was more pronounced and associated with the increased reversible detachment of the cells from the substrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minutes of addition, ethylisopropylamiloride induced pseudopodial retraction and inhibited cell motility. The ability of ethylisopropylamiloride to prevent nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO(3) medium, showing that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesion and pseudopodial protrusion of motile, invasive tumor cells.

Actin Cytoskeleton↗

PubMed1999

Functional expression of tagged human Na+-glucose cotransporter in Xenopus laevis oocytes.

1. High-affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLT1 (sodium glucose cotransporter). Though basic properties of the transporter are now defined, many questions regarding the structure- function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to better address these questions, we produced a functional hSGLT1 protein (from human) containing a reporter tag. 2. Six constructs, made from three tags (myc, haemaglutinin and poly-His) inserted at both the C- and N-terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLT1 construct with the myc tag inserted at the N-terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc-tagged and the untagged hSGLT1 proteins. 3. On the basis of both steady-state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady-state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4. The myc-tagged hSGLT1 was also successfully expressed in polarized MDCK cells. alpha-Methylglucose uptake studies on transfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5. These comparative studies demonstrate that the myc epitope inserted at the N-terminus of hSGLT1 produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.

Animals↗

PubMed1999

Modulation of liver cell membrane NHE-1, Na+-K+ ATPase, and GLUT-2 protein content after cold preservation and rewarming.

Liver cell pH and volume regulation are perturbed by prolonged cold storage in University of Wisconsin solution and subsequent rewarming, but the molecular basis of this effect remains unknown. We prepared membranes from hepatocytes subjected to variable periods of cold preservation with or without subsequent rewarming and probed them by Western blotting with specific antibodies against the Na+ -H+ exchanger isoform NHE-1 and the Na+ -K+ ATPase alpha subunit. Results were compared with the content of GLUT-2, an abundant basolateral protein. NHE-1 decreased significantly as cold preservation times exceeded 10 h. Subsequent rewarming by short-term culture at 37 degrees C did not further reduce this parameter. On the other hand, expression of Na+ -K+ ATPase remained stable during cold storage times lasting up to 48 h, whereas rewarming resulted in a dramatic reduction in cells cold preserved beyond 10 h. In contrast, the membrane content of GLUT-2 was unaffected by cold preservation with or without subsequent rewarming. The results indicate that cold storage and rewarming respectively and selectively modulate the expression of specific hepatocellular membrane transport proteins.

Animals↗

PubMed1998

Zinc cluster proteins Leu3p and Uga3p recognize highly related but distinct DNA targets.

Members of the family of fungal zinc cluster DNA-binding proteins possess 6 highly conserved cysteines that bind to two zinc atoms forming a structure (Zn2Cys6) that is required for recognition of specific DNA sequences. Many zinc cluster proteins have been shown to bind as homodimers to a pair of CGG triplets oriented either as direct (CGG NX CGG), inverted (CGG NX CCG), or everted repeats (CCG NX CGG), where N indicates nucleotides. Variation in the spacing between the CGG triplets also contributes to the diversity of sites recognized. For example, Leu3p binds to the everted sequence CCG N4 CGG with a strict requirement for a 4-base pair spacing. Here, we show that another member of the family, Uga3p, recognizes the same DNA motif as Leu3p. However, these transcription factors have distinct DNA targets. We demonstrate that additional specificity of binding is provided by nucleotides located between the two everted CGG triplets. Altering the 4 nucleotides between to the two everted CGG triplets switches the specificity from a Uga3p site to a Leu3p site in both in vitro and in vivo assays. Thus, our results identify a new mechanism that expands the repertoire of DNA targets of the family of zinc cluster proteins. These experiments provide a model for discrimination between targets of zinc cluster proteins.

Base Sequence↗

PubMed1997

Random mutagenesis reveals a novel site involved in inhibitor interaction within the fourth transmembrane segment of the Na+/H+ exchanger-1.

We constructed and expressed human Na+/H+ exchanger (NHE-1 isoform) cDNAs randomly mutagenized within the sequence encoding the transmembrane region of the exchanger. Using acute intracellular acidifications in the presence of the NHE-1 inhibitor amiloride (300 microM), we selected a clone expressing a NHE-1 protein exhibiting a 3.3-fold increase in K(i) for amiloride (10 microM instead of 3 microM). Sequencing its cDNA revealed one point mutation resulting in a Gly174Ser substitution near the carboxy-terminal end of the putative fourth transmembrane domain of NHE-1. The introduction of this mutation into the wild-type NHE-1 cDNA and its expression reproduced the features of the mutant. Site-directed Gly174Ala and Gly174Asp substitutions resulted, respectively, in no change and in an approximately 4-fold decrease in the amiloride affinity. An additional mutation (Leu163Phe) in transmembrane segment four has previously been shown to result in a decreased sensitivity to amiloride and its derivatives. The Leu163Phe/Gly174Ser double mutant possesses a strongly reduced affinity for various inhibitors (17 microM for amiloride, 2 microM for MPA, and 20 microM for HOE694) and also a decreased affinity (28 mM instead of 14 mM) for sodium. Although distant in the transmembrane segment, Leu163 and Gly174 residues are both not hydrogen-bonded, being one helix turn from proline residues, and are therefore located in highly flexible regions of the protein. This flexibility and the availability of free carbonyls may play an important role in the interaction with the inhibitors and transported cations.

Amiloride↗

PubMed1996

Isolation of heavy endosomes from dog proximal tubules in suspension.

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42-44 kDa and 20-24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as "heavy endosomes" (buoyant density D = 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.

Animals↗

PubMed1995

Cytoplasmic domain of the ubiquitous Na+/H+ exchanger NHE1 can confer Ca2+ responsiveness to the apical isoform NHE3.

The Na+/H+ exchanger isoforms NHE1 and NHE3 are regulated differently by various stimuli. Calcium has been recognized as one of the major second messengers in such exchanger regulation. We previously proposed that Ca(2+)-induced activation of NHE1 occurs via displacement of its autoinhibitory domain from the H+ modifier site due to direct binding of Ca2+/calmodulin. To further validate this hypothesis, the functional role of the cytoplasmic domain was studied in both wild-type and chimeric exchangers, i.e. NHE1, NHE3, NHE1 with the cytoplasmic domain of NHE3 (N1N3), and NHE3 with the cytoplasmic domain of NHE1 (N3N1). After expression in exchanger-deficient fibroblasts (PS120), early response (< 80 s) to external stimuli was assessed as 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Among stimuli tested (ionomycin, alpha-thrombin, phorbol ester, hyperosmotic stress, and platelet-derived growth factor) that are all known to activate NHE1, only ionomycin and thrombin induced a significant intracellular Ca2+ mobilization and early activation of 22Na+ uptake, implying that Ca2+ is a main regulator of NHE1 in the early phase of the agonist response. However, all the stimuli did not activate NHE3 or N1N3. In contrast, a significant stimulation of 22Na+ uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pHi sensitivity (approximately 0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pHi sensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca(2+)-induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H(+)-modifier site of a different isoform, NHE3.

Amiloride↗

PubMed1995

Could cytoplasmic concentration gradients for sodium and ATP exist in intact renal cells?

In renal cells, the Na+ pump maintains a transmembrane concentration gradient for sodium ensuring the net reabsorption of sodium with or without cotransported species. This process requires a significant fraction of the ATP turnover of proximal tubules and thick ascending limbs. To understand the potential regulatory influences of Na+ and ATP on the activity of the Na+ pump in these nephron segments, the apparent kinetics of the membrane-bound Na+-K+ ATPase and of the cellular Na+ pump were studied in different preparations of dog proximal tubules and thick ascending limbs (tubular suspensions, tissue homogenates, and basolateral membrane vesicles) obtained from dog kidney cortex and red medulla. Two determinant kinetic parameters, i.e., the apparent Michaelis constant (Km) and the saturating concentrations for sodium and ATP, were compared with the intracellular concentrations of Na+ and ATP measured under physiological conditions. In both types of tubules, the apparent Km value for Na+ (5-15 mM) is set well below the measured mean intracellular concentration of sodium (50-60 mM), suggesting that the Na+ pump should be saturated by sodium ions under normal conditions. Nevertheless, a modest increment of the Na concentration in the vicinity of the pump, obtained by equilibrating the intra- and extra-cellular sodium concentrations at various extracellular [Na+] with nystatin, increases the activity of the Na+ pump in intact cortical tubules and thick ascending limbs, even when the extracellular [Na+] is set at the estimated intracellular [Na+], demonstrating that the pump is not saturated by sodium in situ. Similarly, the kinetics of the renal Na+ pump as a function of the ATP concentration suggested that the pump should be saturated by ATP in physiological conditions, since in both tissues the cellular ATP level (3-6 mM) is higher than the concentration required to achieve saturation of this activity (< 2.5 mM). However, in renal cortical tubules, the steady-state intracellular [Na+] is affected by modest changes of ATP concentration, suggesting that the Na+ pump is not functionally saturated by ATP. Our data suggest that concentration gradients for Na+ and ATP may exist in the cytosol of renal cells. These gradients would be related to the polarity of sodium transport and of the ATP-consuming and ATP-regenerating processes in intact cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1995

Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms.

Since the cloning of the first member of the Na+/H+ exchanger (NHE) family, termed NHE1, four NHE isoforms have been cloned (NHE2, NHE3, NHE4, and the trout beta-NHE) and expressed in exchanger-deficient cell lines. All these isoforms exhibit significant identity to NHE1 and possess a similar hydropathy profile with two highly conserved transmembrane segments presumably involved in ion transport. These isoforms are allosterically activated by intracellular H+, regulate intracellular pH in a Na(+)-dependent manner, and are inhibited by amiloride and 5-amino derivatives with distinct Ki values. NHE1 is the amiloride-sensitive, growth factor-activatable, and ubiquitously expressed NHE known to regulate intracellular pH and cellular volume. NHE2, NHE3, and NHE4 are, however, restricted in their tissue distribution, suggesting roles in specialized functions of these epithelial tissues. In this review we present and discuss the most recent advances in the molecular and biochemical features, hormonal and growth factor activation, specific expression, and membrane sorting of the members of this NHE family.

Amiloride↗

PubMed1994

Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers.

In this study, we have investigated whether members of the Na+/H+ exchanger (NHE) family are oligomers and whether such oligomeric structure is required for function. Fibroblasts overexpressing NHE1 were treated briefly at 0 degrees C with the cross-linker disuccinimidyl suberate, then membranes were prepared and proteins analyzed by SDS-polyacrylamide gel electrophoresis. Disuccinimidyl suberate treatment converted quantitatively the immunoreactive monomeric form of NHE1 (110 kDa) to a putative dimeric form (210 kDa). Utilization of NHE1 mutant deleted of the cytoplasmic domain (delta 515TH) demonstrates that the transmembrane domain of the antiporter is sufficient for dimerization. Moreover, coimmunoprecipitation of NHE1 and delta 515TH, coexpressed in the same cell, formally proved the existence of dimers. This dimerization was also shown to take place with the epithelial and apically expressed NHE3 isoform, suggesting that oligomerization is a common feature of these transporters. However, coexpression of NHE1 and NHE3 in the same cells did not lead to the formation of heterodimers demonstrating an isoform specificity for the subunit interaction. The domain(s) involved in the isoform-specific dimerization is (are) likely to be confined within the transmembrane segments, as deletion of the 300 amino acids of the cytoplasmic domain did not disrupt dimerization. Exploiting the dimeric properties of the receptor tyrosine kinases and the fact that dimerization triggers kinase activity, we constructed a NHE1/insulin receptor chimera to probe NHE1 dimerization in vivo. When transfected into hamster fibroblasts, this chimera containing the N-terminal transmembrane domain of NHE1 and the cytoplasmic beta-subunit domain of the insulin receptor generates a functional transporter that is autophosphorylated on tyrosine and that presents properties of a constitutively active insulin receptor. These findings support the notion that NHE1 exists in an oligomeric state in intact cells. Finally, to test whether individual subunits of NHE1 are the minimum functional unit for Na+/H+ exchange, we coexpressed a truncated form of NHE1 (delta 515) together with an inactive mutant of NHE1 (E262I). In spite of good expression of the inactive transporter and its capacity to dimerize with active NHE1, no dominant negative effect was observed on amiloride-sensitive 22Na+ flux. This observation would suggest that individual subunits of NHE1 function independently within the oligomeric state.

Animals↗