Biomedical subjects

F Verrey

Publications and source records attributed to F Verrey.

At least 19 recordsLinked to original sources

PubMed2001

Short term effect of aldosterone on Na,K-ATPase cell surface expression in kidney collecting duct cells.

Aldosterone controls extracellular volume and blood pressure by regulating Na+ reabsorption, in particular by epithelia of the distal nephron. A main regulatory site of this transcellular transport is the epithelial sodium channel (ENaC) that mediates luminal Na+ influx. The Na,K-ATPase (Na+ pump) that coordinately extrudes Na+ across the basolateral membrane is known to be regulated by short term aldosterone as well. We now show that in the cortical collecting duct (CCD) from adrenalectomized rats, the increase in Na,K-ATPase activity (approximately 3-fold in 3 h), induced by a single aldosterone injection, can be fully accounted by the increase in Na,K-ATPase cell surface expression (+ 497 +/- 35%). The short term aldosterone action was further investigated in cultured mouse collecting duct principal cells mpkCCD(cl4). Within 2 h, maximal Na,K-ATPase function assessed by Na+ pump current (I(p)) measurements and Na,K-ATPase cell surface expression were increased by 20-50%. Aldosterone did not modify the Na+ dependence of the Na+ pumps and induced transcription- and translation-dependent actions on pump surface expression and current independently of ENaC-mediated Na+ influx. In summary, short term aldosterone directly increases the cell surface expression of pre-existing Na+ pumps in kidney CCD target cells. Thus, aldosterone controls Na+ reabsorption in the short term not only by regulating the apical cell surface expression of ENaC (Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B. C., Firestone, G. L., Pearce, D., and Verrey, F. (2001) Am. J. Physiol. 280, F675-F682) but also by coordinately acting on the basolateral cell surface expression of the Na,K-ATPase.

Aldosterone↗

PubMed2001

Sodium reabsorption in aldosterone-sensitive distal nephron: news and contributions from genetically engineered animals.

The precise adaptation of renal sodium excretion to systemic needs is to a large extent achieved by the regulation of sodium re-absorption in the aldosterone-sensitive distal nephron. Transcellular sodium re-absorption by the segment-specific cells of the aldosterone-sensitive distal nephron (often called principal cells) is mainly controlled at the level of the expression and activity levels of the epithelial sodium channel, the apical amiloride-sensitive sodium influx pathway. Recent investigations have identified the first early aldosterone-induced proteins that act on epithelial sodium channel function in expression systems. Indirect evidence suggests that one of these aldosterone-induced proteins, the serum- and glucocorticoid-inducible protein kinase SGK1, plays a central integratory role in the control of epithelial sodium channel surface expression and activity, also in the mammalian kidney. Gene-modified animals lacking epithelial sodium channel subunits or expressing mutant subunits have substantiated the central role of the epithelial sodium channel in sodium re-absorption and blood pressure control, as well as for neonatal lung liquid clearance. Mice overexpressing or lacking specific hormones or their receptors have been used to study their role in sodium transport regulation, but the study of mouse physiology appears to lag behind the generation of gene-modified mice. Nonetheless, these new animal models have had a strong impact on research, by stimulating the integration of knowledge and techniques learned from reductionistic molecular approaches into tissue and animal studies, thus breaking down barriers and stimulating collaborations.

Absorption↗

PubMed2001

Mediators of aldosterone action in the renal tubule.

The aldosterone-sensitive distal nephron extends from the second part of the distal convoluted tubule to the inner medullary collecting duct. As recently shown, aldosterone increases within two hours the abundance of the alpha-subunit of the epithelial sodium channel along the entire aldosterone-sensitive distal nephron, whereas it induces only in an initial portion of the aldosterone-sensitive distal nephron an apical translocation of all three epithelial sodium channel subunits. This suggests that another factor or factors determines the length of the aldosterone-sensitive distal nephron portion in which aldosterone controls epithelial sodium channel surface expression. Since the glucocorticoid-induced kinase SGK1 was identified as aldosterone-induced protein in 1999, it has been postulated to play a key regulatory role. The in-vivo localization of its induction to segment-specific cells of the aldosterone-sensitive distal nephron, and the in-vitro correlation of the amount of its hyperphosphorylated form with transepithelial sodium transport, support this hypothesis. Other recent studies unravel pathways other than those activated by aldosterone and insulin that impact on SGK1 expression and/or function, and thus shed some light onto the complex network that appears to control sodium transport. In view of the ongoing research, the question of how, and formally also whether, SGK1 acts on the epithelial sodium channel should be resolved in the near future.

Aldosterone↗

PubMed2001

Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK.

Aldosterone controls sodium reabsorption and potassium secretion in the aldosterone-sensitive distal nephron (ASDN). Although clearance measurements have shown that aldosterone induces these transports within 30--60 min, no early effects have been demonstrated in vivo at the level of the apical epithelial sodium channel (ENaC), the main effector of this regulation. Here we show by real-time RT-PCR and immunofluorescence that an aldosterone injection in adrenalectomized rats induces alpha-ENaC subunit expression along the entire ASDN within 2 h, whereas beta- and gamma-ENaC are constitutively expressed. In the proximal ASDN portions only, ENaC is shifted toward the apical cellular pole and the apical plasma membrane within 2 and 4 h, respectively. To address the question of whether the early aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) might mediate this apical shift of ENaC, we analyzed SGK induction in vivo. Two hours after aldosterone, SGK was highly induced in all segment-specific cells of the ASDN, and its level decreased thereafter. In Xenopus laevis oocytes, SGK induced ENaC activation and surface expression by a kinase activity-dependent mechanism. In conclusion, the rapid in vivo accumulation of SGK and alpha-ENaC after aldosterone injection takes place along the entire ASDN, whereas the translocation of alpha,beta,gamma-ENaC to the apical plasma membrane is restricted to its proximal portions. Results from oocyte experiments suggest the hypothesis that a localized activation of SGK may play a role in the mediation of ENaC translocation.

Aldosterone↗

PubMed2001

Thyroid hormone transport by the heterodimeric human system L amino acid transporter.

Transport of thyroid hormone across the cell membrane is required for thyroid hormone action and metabolism. We have investigated the possible transport of iodothyronines by the human system L amino acid transporter, a protein consisting of the human 4F2 heavy chain and the human LAT1 light chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2 heavy chain and/or human LAT1 light chain, and after 2 d were incubated at 25 C with 0.01-10 microM [(125)I]T(4), [(125)I]T(3), [(125)I]rT(3), or [(125)I]3,3'-diiodothyronine or with 10-100 microM [(3)H]arginine, [(3)H]leucine, [(3)H]phenylalanine, [(3)H]tyrosine, or [(3)H]tryptophan. Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of leucine and arginine due to dimerization of human 4F2 heavy chain with an endogenous Xenopus light chain, but did not affect the uptake of other ligands. Injection of human LAT1 light chain cRNA alone did not stimulate the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain and human LAT1 light chain stimulated the uptake of phenylalanine > tyrosine > leucine > tryptophan (100 microM) and of 3,3'-diiodothyronine > rT(3) approximately T(3) > T(4) (10 nM), which in all cases was Na(+) independent. Saturation analysis provided apparent Michaelis constant (K(m)) values of 7.9 microM for T(4), 0.8 microM for T(3), 12.5 microM for rT(3), 7.9 microM for 3,3'-diiodothyronine, 46 microM for leucine, and 19 microM for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 microM) was inhibited by the different iodothyronines (10 microM), in particular T(3). Vice versa, uptake of 0.1 microM T(3) was almost completely blocked by coincubation with 100 microM leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na(+)-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter.

Amino Acids↗

PubMed2000

Glycoprotein-associated amino acid exchangers: broadening the range of transport specificity.

Members of the newly discovered glycoprotein-associated amino acid transporter family (gpaAT-family) share a similar primary structure with >40% identity, a predicted 12-transmembrane segment topology and the requirement for association with a glycoprotein (heavy chain) for functional surface expression. Five of the six identified gpaATs (light chains) associate with the surface antigen 4F2 heavy chain (4F2hc = CD98), a ubiquitous plasma membrane protein induced in cell proliferation, and which is also highly expressed at the basolateral surface of amino acid transporting epithelia. The differing tissue localizations of the 4F2hc-associated gpaATs appear to complement each other. As yet, a single gpaAT (b(0,+)AT) has been shown to associate with rBAT, a 4F2hc-related glycoprotein mainly localized in intestine and kidney luminal brush-border membranes. The transport characteristics of gpaATs have been shown, by expression in heterologous systems, to correspond to the previously described transport systems L, y+L, xc- and b(o,+). These (obligatory) exchangers of broad substrate specificity (with the exception of xCT) are expected to equilibrate the concentrations of their substrate amino acids across membranes. Thus, the driving force provided by a transmembrane gradient of one substrate amino acid, such as that generated by a parallel functioning unidirectional transporter, can be used by a gpaAT to fuel the secondary active vectorial transport of other exchangeable species. Vectorial transport of specific amino acids is also promoted by the intrinsic asymmetry of these exchangers. The fact that genetic defects of the epithelial gpaATs b(0,+)AT and y+LAT1 cause non-type I cystinuria and lysinuric protein intolerance, respectively, demonstrates that these gpaATs perform vectorial secondary and/or tertiary active transport of specific amino acids in vivo.

Amino Acid Transport Systems↗

PubMed2000

cAMP sensitivity conferred to the epithelial Na+ channel by alpha-subunit cloned from guinea-pig colon.

The rate of Na+ (re)absorption across tight epithelia such as in distal kidney nephron and colon is to a large extent controlled at the level of the epithelial Na+ channel (ENaC). In kidney, antidiuretic hormone (ADH, vasopressin) stimulates the expression/activity of this channel by a cAMP/protein-kinase-A- (PKA-) mediated pathway. However, a clear upregulation of ENaC function by cAMP could not be reproduced with cloned channel subunits in the Xenopus oocyte expression system, suggesting the hypothesis that an additional factor is missing. In contrast, we show here that membrane-permeant cAMP can activate ENaC expressed in Xenopus oocytes (3.8-fold) upon replacement of the rat alpha-subunit by a new alpha-subunit cloned from guinea-pig colon (gpalpha). This alpha-subunit is 76% identical with its rat orthologue originating from ADH-insensitive rat colon. The biophysical fingerprints of the hybrid ENaC formed by this guinea-pig alpha-subunit together with rat beta- and gamma-subunits are indistinguishable from those of rat ENaC (rENaC). Injection of the PKA inhibitor PKI-(6-22)-amide into the oocyte had no effect on the basal activity of rat ENaC but inhibited the activity of gpalpha-containing hybrid ENaC and greatly decreased its stimulation by cAMP. This suggests that, unlike for rat ENaC, tonic PKA activity is required for basal function of gpalpha-containing ENaC and that PKA mediates its cAMP-induced activation. This regulatory behaviour is not common to all ENaCs containing an alpha-subunit cloned from an ADH-responsive tissue since xENaC, which was cloned from the ADH-sensitive Xenopus laevis A6 epithelia, is, when expressed in oocytes, resistant to cAMP, similar to rat ENaC. This study demonstrates that the PKA sensitivity of ENaC can depend on the nature of the ENaC alpha-subunit and raises the possibility that cAMP can stimulate ENaCs by different mechanisms.

Amiloride↗

PubMed2000

Introduction

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Journal Article↗

PubMed2000

Pleiotropic action of aldosterone in epithelia mediated by transcription and post-transcription mechanisms.

The aldosterone-induced increase in sodium reabsorption across tight epithelia can be divided schematically into two functional phases: an early regulatory phase starting after a lag period of 20 to 60 minutes, during which the pre-existing transport machinery is activated, and a late phase (>2.5 h), which can be viewed as an anabolic action leading to a further amplification/differentiation of the Na+ transport machinery. At the transcriptional level, both early and late responses are initiated during the lag period, but the functional impact of newly synthesized regulatory proteins is faster than that of the structural ones. K-Ras2 and SGK were identified as the first early aldosterone-induced regulatory proteins in A6 epithelia. Their mRNAs also were shown to be regulated in vivo by aldosterone, and their expression (constitutively active K-Ras2 and wild-type SGK) was shown to increase the function of ENaC coexpressed in Xenopus oocytes. Recently, aldosterone was also shown to act on transcription factors in A6 epithelia: It down-regulates the mRNAs of the proliferation-promoting c-Myc, c-Jun, and c-Fos by a post-transcriptional mechanism, whereas it up-regulates that of Fra-2 (c-Fos antagonist) at the transcriptional level. Together, these new data illustrate the complexity of the regulatory network controlled by aldosterone and support the view that its early action is mediated by the induction of key regulatory proteins such as K-Ras2 and SGK. These early induced proteins are sites of convergence for different regulatory inputs, and thus, their aldosterone-regulated expression level tunes the impact of other regulatory cascades on sodium transport. This suggests mechanisms for the escape from aldosterone action.

Aldosterone↗

PubMed2000

Role of SGK in mineralocorticoid-regulated sodium transport.

Mineralocorticoids stimulate electrogenic Na+ transport in tight epithelia by altering the transcription of specific genes. Although the earliest mineralocorticoid effect is to increase the activity of the epithelial sodium channel (ENaC), ENaC mRNA and protein levels do not change. Instead, physiologic observations suggest that a mineralocorticoid target gene(s) encodes an ENaC regulator(s). To begin to identify and characterize mineralocorticoid-regulated target genes, we used suppression-subtractive hybridization to generate a cDNA library from A6 cells, a stable cell line of Xenopus laevis of distal nephron origin. A serine-threonine kinase, SGK, was identified from this screen. Sequence comparison revealed that frog, rat, and human SGK are 92% identical and 96% similar at the amino acid level. SGK mRNA was confirmed by Northern blot to be strongly and rapidly corticosteroid stimulated in A6 cells. In situ hybridization revealed that SGK was strongly stimulated by aldosterone in rat collecting duct but not proximal tubule cells. Low levels of SGK were present in rat glomeruli, but SGK was unregulated in this structure. Finally, SGK stimulated ENaC activity approximately sevenfold when coexpressed in Xenopus laevis oocytes. These data suggest that SGK is an important mediator of aldosterone effects on Na+ transport in tight epithelia. In view of the existence of SGK homologues in invertebrates, it is interesting to speculate that SGK is an ancient kinase that was adapted to the control of epithelial Na+ transport by early vertebrates as they made the transition from a marine to a freshwater environment.

Amino Acid Sequence↗

PubMed1999

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine.

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.

Amino Acid Transport Systems↗

PubMed1999

New glycoprotein-associated amino acid transporters.

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked "light chain" of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y(+)LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b(0,+)AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family).

Amino Acid Transport System y+↗

PubMed1999

Regulation of Na+ pump function by aldosterone is alpha-subunit isoform specific.

1. During its early 'genomic' phase of action (< 3 h), aldosterone activates pre-existing Na+ pumps (Na+,K+-ATPase) in epithelia formed by Xenopus laevis A6 kidney cells. 2. To test whether this action also applies to pumps containing mammalian alpha-subunits of different isoforms, we generated A6 cell lines expressing the naturally ouabain-resistant rat alpha1 subunit or the rat alpha2* and alpha3* subunits made ouabain resistant by site-directed mutagenesis. 3. Cell lines were obtained which expressed the exogenous alpha-subunits in active, basolateral Na+ pumps, such that ouabain-resistant pump current (Ip) could be measured following apical permeabilization with amphotericin B. 4. The inhibition constants (Ki) for ouabain of the current carried by the pumps containing exogenous rat alpha-subunits were similar to those reported previously for ATPase activity inhibition. The apparent Michaelis constant (Km) for Na+ (K+ replacement) was slightly higher for pumps containing the rat alpha1 than for those containing the alpha2* subunit (34.9 +/- 1.9 versus 26.3 +/- 2.6 mM). 5. At a Na+ concentration of 10 mM, aldosterone (2.5 h) increased the pump current carried by endogenous pumps as well as that carried by pumps containing the exogenous rat alpha1 subunit (by 1.8- to 2.2-fold). In contrast, the current carried by pumps containing the exogenous rat alpha2* subunit remained unchanged. 6. The fact that this early transcriptionally mediated activation of Na+ pumps by aldosterone is specific for pumps containing an alpha1 subunit should permit the identification in this subunit of structures involved in its regulation.

Aldosterone↗

PubMed1999

Adenosine inhibits the transfected Na+-H+ exchanger NHE3 in Xenopus laevis renal epithelial cells (A6/C1).

1. Adenosine influences the vectorial transport of Na+ and HCO3- across kidney epithelial cells. However, its action on effector proteins, such as the Na+-H+ exchanger NHE3, an epithelial brush border isoform of the Na+-H+ exchanger (NHE) gene family, is not yet defined. 2. The present study was conducted in Xenopus laevis distal nephron A6 epithelia which express both an apical adenosine receptor of the A1 type (coupled to protein kinase C (PKC)) and a basolateral receptor of the A2 type (coupled to protein kinase A (PKA)). The untransfected A6 cell line expresses a single NHE type (XNHE) which is restricted to the basolateral membrane and which is activated by PKA. 3. A6 cell lines were generated which express exogenous rat NHE3. Measurements of side-specific pHi recovery from acid loads in the presence of HOE694 (an inhibitor with differential potency towards individual NHE isoforms) detected an apical resistant Na+-H+ exchange only in transfected cell lines. The sensitivity of the basolateral NHE to HOE694 was unchanged, suggesting that exogenous NHE3 was restricted to the apical membrane. 4. Stimulation of the apical A1 receptor with N 6-cyclopentyladenosine (CPA) inhibited both apical NHE3 and basolateral XNHE. These effects were mimicked by the addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and partially prevented by the PKC inhibitor calphostin C which also blocked the effect of PMA. 5. Stimulation of the basolateral A2 receptor with CPA inhibited apical NHE3 and stimulated basolateral XNHE. These effects were mimicked by 8-bromo-cAMP and partially prevented by the PKA inhibitor H89 which entirely blocked the effect of 8-bromo-cAMP. 6. In conclusion, CPA inhibits rat NHE3 expressed apically in A6 epithelia via both the apical PKC-coupled A1 and the basolateral PKA-coupled A2 adenosine receptors.

Adenosine↗

PubMed1999

Epithelial sodium channel regulated by aldosterone-induced protein sgk.

Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na+ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na+ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na+ absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis.

Aldosterone↗

PubMed1999

Amino acid transport of y+L-type by heterodimers of 4F2hc/CD98 and members of the glycoprotein-associated amino acid transporter family.

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na+-independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease-related protein (LAT1 or AmAT-L-lc) which for surface expression and function requires formation of disulfide-linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y+LAT1 from mouse and human which are approximately 48% identical with LAT1 and thus belong to the same family of glycoprotein-associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na+-dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmacological fingerprints identify them as y+L-type transport systems. The mRNA encoding my+LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y+LAT1-4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in transepithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance.

Amino Acid Sequence↗

PubMed1999

SPRM1lc, a heterodimeric amino acid permease light chain of the human parasitic platyhelminth, Schistosoma mansoni.

The Schistosoma mansoni protein, SPRM1lc, is a light chain member of a new family of heterodimeric amino acid permeases. These proteins require covalent association with a type II glycoprotein (like h4F2hc) for functional surface localization when expressed in Xenopus oocytes. We previously reported that, when co-expressed with h4F2hc, the transport properties of SPRM1lc resemble system y and y+ while its human homologue, E16, functions as an L-type permease. Here we extend the functional characterization of SPRM1lc in oocytes and show by competitor studies that its amino acid transport capacity is similar to that of whole adult schistosomes. We demonstrate by Northern and Western analysis that SPRM1lc is expressed within both larval and adult schistosomes. In all stages, SPRM1lc is associated into a high molecular weight complex that can be disrupted by reducing agents, consistent with the hypothesis that a significant fraction of the endogenous SPRM1lc is linked by a disulphide bond to an uncharacterized schistosome amino acid transporter heavy chain. Immunofluorescence localization detects SPRM1lc in miracidia, daughter sporocysts and adult worms. Confocal microscopy demonstrates that SPRM1lc is found in the apical membrane of the syncytial, double-lipid bilayer tegument which surrounds adult worms. Aqueous biotinylation studies on living worms show that SPRM1lc is exposed on the host-interactive surface of this tegumental membrane. Host exposed, functionally important surface proteins such as SPRM1lc could form the basis of an effective schistosomiasis vaccine. These studies are the first to describe a helminth amino acid transporter, and the first to characterize an invertebrate heterodimeric amino acid transporter.

Amino Acid Sequence↗

PubMed1999

Luminal heterodimeric amino acid transporter defective in cystinuria.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.

Amino Acid Sequence↗