Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (Plenary Lecture)

Renal and small intestinal (re-)absorption contribute to overall phosphate(P_i)-homeostasis. In both epithelia, apical sodium (Na⁺)/P_i-cotransport across the luminal (brush border) memi brane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/P_i-cotransporters have been identified: (i) type I cotransporter(s) - present in the proximal tubule - also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type IIa)and in the small intestine (type IIb), isoform determines Na⁺-dependent transcellular P_i-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in P_i-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered P_i-intake, levels of 1.25 (OH₂) vitamin D₃, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/P_i-cotransporters operate (as functional monomers) in a 3 Na⁺:1 P_i stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na⁺, slippage)and of the fully loaded carriers (3 Na⁺, 1 P_i). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na⁺-interaction or specific pH-dependence of the IIa and IIb cotransporters. For the COOH-terminal tail of the IIa Na/P_i -cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).     

Cleavage of Disulfide Bonds Leads to Inactivation and Degradation of the Type IIa, But not Type IIb Sodium Phosphate Cotransporter Expressed in Xenopus laevis Oocytes

Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter (rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/P _i -cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with TCEP the function of NaPi IIa was inhibited and protein amount was decreased. Injection of the lysosomal inhibitor leupeptin prevented degradation of the protein. Exposure of oocytes to TCEP at 0°C led to a reduction in transport function without concomitant loss in Na/Pi IIa protein. In contrast to NaPi type IIa, the type IIb isoform was neither inhibited, nor degraded after incubation with TCEP. These results suggest that cleavage of disulfide bonds led to changes within the confirmation of the type IIa transporter that result in (i) inhibition of the transport activity and (ii) internalization and subsequent lysosomal degradation of transporter protein. Sequence comparisons suggest the involvement/presence of different disulfide bonds in type IIa and type IIb Na/P _i -cotransporters. Received: 13 December 1999/Revised: 31 March 2000     

Functional Expression and Purification of Histidine-Tagged Rat Renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) Cotransporters

Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/P _i -cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/P _i -cotransport exhibited a K _m of P _i of 0.21 mm and an apparent K _m of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a K _m of SO²⁻ ₄ of about 0.3–0.4 mm and a K _m of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaS _i -1 proteins were detected at 55–60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography. Received: 24 March 1997/Revised: 8 July 1997     

Functional expression of rat renal Na/Pi-cotransport (NaPi-2) in Sf9 cells by the baculovirus system

The recently cloned Na/P _i -cotransport system NaPi-2 is an apical membrane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further functional/structural studies, this transport system has been expressed in Sf9 insect cells using a recombinant baculovirus. Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/P _i -cotransport up to 20- to 50-fold over noninfected Sf9 cells. Transport of phosphate in infected cells was highly dependent on sodium, exhibited a K _m for P _i of 0.114 mm and an apparent K _m for Na of 63 mm (Hill coefficient of approximately 3) and was stimulated by high external pH. Infected cells expressed a polypeptide of 65 kDa representing a nonglycosylated form of the 85 kDa mature NaPi-2 transporter as present in proximal tubular brush-border membranes. By confocal microscopy expression of NaPi-2 protein was observed in the plasma membrane, yet submembranous accumulation of NaPi-2 protein could not be excluded. This demonstrates that the rat proximal tubular Na/P _i -cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics similar to that in isolated brush-border membranes. The 6His tagging will permit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.We would like to thank W. Scherle and M. Lötscher (Institute of Anatomy) for their generous help using the confocal microscope and Ch. Gasser for the art work. Financial support by the Swiss National Fonds [Grant No. 32-30785.91 (to H.M.) and 32-28664.90 (to J.B.)] and by Stiftung für wissenschaftliche Forschung an der Universitát Zürich is greatly acknowledged.     

The renal type IIa Na/Pi cotransporter: structure-function relationships

The type IIa Na/P_i cotransporter mediates proximal tubular brush-border membrane secondary active phosphate (P_i) flux. It is rate limiting in tubular P_i reabsorption and, thus, a final target in many physiological and pathophysiological situations of altered renal P_i handling (1–4). In the present short review, we will briefly summarize our current knowledge about the transport mechanism (cycle) as well as particular regions of the transporter protein (“molecular domains”) that potentially determine transport characteristics.     

Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (plenary lecture)

Renal and small intestinal (re-)absorption contribute to overall phosphate(Pi)-homeostasis. In both epithelia, apical sodium (Na+)/Pi-cotransport across the luminal (brush border) membrane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/Pi-cotransporters have been identified: (i) type I cotransporter(s)--present in the proximal tubule--also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type Ila) and in the small intestine (type IIb), isoform determines Na+-dependent transcellular Pi-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in Pi-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered Pi-intake, levels of 1.25 (OH2) vitamin D3, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/Pi-cotransporters operate (as functional monomers) in a 3 Na+:1 Pi stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na+, slippage) and of the fully loaded carriers (3 Na+, 1 Pi). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na+-interaction or specific pH-dependence of the IIa and IIIb cotransporters. For the COOH-terminal tail of the IIa Na/Pi-cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).     

Effect of Two Tyrosine Mutations on the Activity and Regulation of the Renal Type II Na/P i -Cotransporter Expressed in Oocytes

The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY₄₀₂FAM matching the consensus sequence GYXXZ, and Y₅₀₉RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated ³²P _i -uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/P _i -cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter. Received: 3 November 1998/Revised: 20 January 1999     

Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions

Wepreviously demonstrated that inhibition of Na-dependent phosphate(P_i) transport in osteoclasts led to reduced ATP levels anddiminished bone resorption. These findings suggested that Na/P_i cotransporters in the osteoclast plasma membraneprovide P_i for ATP synthesis and that the osteoclast mayutilize part of the P_i released from bone resorption forthis purpose. The present study was undertaken to define the cellularlocalization of Na/P_i cotransporters in the mouseosteoclast and to identify the proteins with which they interact. Usingglutathione S-transferase (GST) fusion constructs, wedemonstrate that the type IIa Na/P_i cotransporter (Npt2a)in osteoclast lysates interacts with the Na/H exchanger regulatoryfactor, NHERF-1, a PDZ protein that is essential for the regulation ofvarious membrane transporters. In addition, NHERF-1 in osteoclastlysates interacts with Npt2a in spite of deletion of a putativePDZ-binding domain within the carboxy terminus of Npt2a. In contrast,deletion of the carboxy-terminal TRL amino acid motif of Npt2asignificantly reduced its interaction with NHERF-1 in kidney lysates.Studies in osteoclasts transfected with green fluorescent protein-Npt2aconstructs indicated that Npt2a colocalizes with NHERF-1 and actin ator near the plasma membrane of the osteoclast and associates withezrin, a linker protein associated with the actin cytoskeleton, likelyvia NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNAand in situ hybridization that the type III Na/P_icotransporter, PiT-1, is also expressed in mouse osteoclasts. Toexamine the cellular distribution of PiT-1, we infected mouseosteoclasts with a retroviral vector encoding PiT-1 fused to an epitopetag. PiT-1 colocalizes with actin and is present on the basolateralmembrane of the polarized osteoclast, similar to that previouslyreported for Npt2a. Taken together, our data suggest that associationof Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, maybe responsible for membrane sorting and regulation of theseNa/P_i cotransporters in the osteoclast.

     

Functionally Important Residues in the Predicted 3<Superscript>rd</Superscript> Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

The type IIa Na⁺/P_i, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na⁺ and one divalent P_i ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P_i transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P_i transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P_i-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na⁺-dependent P_i transport. Since electrogenic NaPi-II transports 3 Na⁺/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na⁺ binding sites in NaPi-IIa.     

Interaction of the type IIa Na/Pi cotransporter with PDZ proteins

The type IIa Na(+)-dependent inorganic phosphate (Na/P(i)) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P(i) cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.     

Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.

We have used baculovirus (AcNPV) to express the Na+/glucose cotransporter protein in cultured Sf9 cells. We constructed a baculovirus transfer vector containing the cDNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) under the control of the polyhedrin gene promoter. Recombinant baculovirus was obtained by cotransfection of SF9 cells with wild-type AcNPV DNA and the transfer vector. Recombinant virus was identified by Southern blotting and then purified. Recombinant infected Sf9 cells expressed a protein which was recognized by anti-peptide antibodies raised to sequences of the cloned Na+/glucose cotransporter. This protein migrated with a molecular mass of 55 kD by SDS-PAGE, similar to the in vitro translation product of SGLT1. An identical protein was metabolically labeled with [35S]methionine. Cells which synthesized the transport protein showed Na(+)-dependent alpha MeGlc transport. Micromolar phlorizin inhibited transport. Uninfected and wild-type virus infected Sf9 cells did not have Na(+)-dependent glucose transport. All transport protein migrated at 45% sucrose (w/w) by density gradient sedimentation, suggesting that the expressed transporter is membrane associated. We conclude that we have functionally expressed the rabbit intestinal Na+/glucose cotransporter in Sf9 cells. The transporter is not heavily glycosylated, and this is consistent with previous work showing that glycosylation is not necessary for function. We are poised to purify and characterize this protein from a structure-function perspective.     

Cysteine Residues and the Structure of the Rat Renal Proximal Tubular Type II Sodium Phosphate Cotransporter (Rat NaPi IIa)

The rat renal Na/P _i cotransporter type IIa (rat NaP_i IIa) is a 637 amino acid protein containing 12 cysteine residues. We examined the effect of different cysteine modifying methanethiosulfonate (MTS)-reagents and the disulfide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transport activity of wild-type and 12 single cysteine substitution mutants of rat NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethiosulfonate (MMTS) and TCEP inhibited the transport activity of both the wild-type, as well as all the single mutant proteins. This indicated the existence of more than one functionally important cysteine residue, not accessible extracellularly, and at least 2 disulfide bridges. To identify the disulfide bridges, three double mutants lacking 2 of the 3 cysteine residues predicted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; reconsideration of the topological model predictions suggested a second disulfide bridge between C225 and C520. Evaluation of a fourth double mutant indicated that at least one of two disulfide bridges (C306 and C334; C225 and C520) has to be formed to allow the surface expression of a functional cotransporter. A revised secondary structure is proposed which includes two partially repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520. Received: 13 December 1999/Revised: 31 March 2000     

Expression of Na/Pi cotransport from opossum kidney cells in Xenopus laevis oocytes

Xenopus laevis oocytes have been used for the expression of Na/P_i-cotransport activity by injections of poly(A)⁺ RNA (mRNA) isolated from an established renal cell line (OK cells). 3–5 days after mRNA injection, Na-dependent phosphate (P_i) uptake by oocytes was increased in a dose-dependent manner; there was no increase in Na-independent P_i uptake. Sucrose density-gradient fractionation indicated that the mRNA species encoding this activity is 2.4–2.8 kb in length. In Northern blots, using a cDNA probe related to human kidney-cortex Na/P_i-cotransport activity (NaPi-3), hybridization with a mRNA-species of 2.4–2.6 kb was obtained. Kinetic characterization ([P_i], [Na]) showed that expressed transport activity has properties similar to apical Na/P_i cotransport in OK cells.     

Molecular determinants of pH sensitivity of the type IIa Na/P(i) cotransporter

Type II Na/P(i) cotransporters play key roles in epithelial P(i) transport and thereby contribute to overall P(i) homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/P(i) cotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/P(i) cotransporter.     

Synapsin IIa: expression in insect cells, purification, and characterization.

Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.     

Synthesis and Biological Activity of 6-Hydroxyguanidino-and 6-Hydroxyureidopurine And Their Ribonucleosides

Abstract

N ⁶ ?(1-hydroxyguanidino)purine IIa, and its 9-β-D-ribonucleoside derivative IIb were prepared by reacting at room temperature 6-hydroxyadenine Ia and 6-hydroxyadenosine Ib, with 1-guanyl-3,5-dimethylpyrazole nitrate in DMF. Refluxing IIa and IIb in 95% ethanol gave N⁶?(1-hydroxyureido)purine and its ribonucleoside derivative respectively; the latter compound was also obtained by refluxing Ib with 1-guanyl-3,5-dimethylpyrazole nitrate in ethanol. The two base analogs were inactive against L1210 cells in vitro, but the nucleoside derivatives inhibited the growth of these cells by 50% at 5 × 10 ^-6 and 6 × 10^?7 M respectively. Compound IIb, at 200 mg/kg/day × 5, increased the life span of L1210-bearing DBA/2N mice by 57%. Cytofluorometric determinations showed that IIb inhibited cell growth in the G₂ phase of the cell cycle. also found to inhibit adenosine deaminase activity with a K_i = 3.47 μM.     

Investigating the Surface Expression of the Renal Type IIa Na+/P i -Cotransporter in Xenopus laevis Oocytes

We have combined a functional assay, surface labeling and immunocytochemical methods to compare total and surface-exposed renal type IIa Na⁺/P _i cotransporter protein. The wild-type type cotransporter (NaPi-IIa) and its functionally comparable cysteine mutant S460C were expressed in Xenopus oocytes. S460C contains a novel cysteine residue that, when modified by preincubation with methanethiosulfonate reagents, leads to complete suppression of cotransport function. This allowed surface labeling of the S460C using MTSEA-Biotin and confirmation by electrophysiology on the same cell. Protein was analyzed by Western blotting before and after streptavidin precipitation and by immunocytochemistry and immunogold electronmicroscopy. MTSEA-Biotin treatment resulted in a complete inhibition of S460C-mediated Na⁺/P _i -cotransport activity, which indicated that all transporters at the surface were biotinylated. After biotinylation, only a small fraction of total S460C protein was precipitated by streptavidin compared with the total amount of S460C protein detected in the lysate. Light- and electron-microscopy analysis of oocytes showed a large amount of WT and S460C transporter protein beneath the oocyte membrane. These data indicate that the apparent weak labeling efficiencies of surface-biotinylation-based assays of membrane proteins heterologously expressed in oocytes can be related to diminished incorporation of the protein in the oolemma. Received: 18 August 2000/Revised: 1 December 2000     

Regulation of intracellular pH by electrogenic Na+/HCO3– co-transporters in embryonic neural stem cell-derived radial glia-like cells

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pH_i) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extracellular K⁺ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extracellular calcium was removed, an instant depolarization and recovery of the pH_i, back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by S0859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pH_i recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pH_i regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pH_i regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.     

Expression of Functional Human Muscarinic M2 Receptors in Different Insect Cell Lines

Abstract

Human M2 receptors were expressed using the baculovirus expression system in three different insect cell lines: Sf9, Sf21 and High5. The level of expression was slightly increased in Sf21 cells versus Sf9 cells. In contrast, High5 cells were not able to produce more recombinant protein than Sf9. We also show that in both Spodoptera frugiperda cell lines a peak of expression was reached after 6 days of infection, whereas in High5 cells, the maximum of expression occurred after 3 days. Immunodetection of m2 muscarinic receptor clearly shows that the expressed protein undergoes significant proteolysis in both the Sf9 and High5 cells, whereas in the Sf21 cells this phenomenon was less detectable. Additionally, we show that in all three cell lines, the expressed recombinant receptor was functional in that it was able to stimulate GTPγS binding in the presence of exogenous G-proteins. Analysis of the population of G-proteins (Gαi₁ Gα_o and Gβcommon) in Sf21 and High5 cells is provided.