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1.
The rat renal Na/P i cotransporter type IIa (rat NaPi 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  相似文献   

2.
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  相似文献   

3.
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  相似文献   

4.
In mammals the type IIb Na/Pi-cotransporter is expressed in various tissues such as intestine, brain, lung and testis. The type IIb cotransporter shows 51% homology with the renal type IIa Na/Pi-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/Pi-cotransporter available for future structural studies, we have expressed this cotransporter in Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing 6His NaPi-IIb. Infected cells expressed a polypeptide of ~90 kDa, corresponding to a partially glycosylated form of the type IIb cotransporter. Transport studies demonstrated that the type IIb protein expressed in Sf9 cells mediates transport of phosphate in a Na-dependent manner with similar kinetic characteristics (apparent K ms for sodium and phosphate and pH dependence) as previously described. Solubilization experiments demonstrated that, in contrast to the type IIa cotransporter, the type IIb can be solubilized by nonionic detergents and that solubilized type IIb Na/Pi-cotransporter can be purified by Ni-NTA chromatography.  相似文献   

5.
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) memi brane 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 IIa)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 IIb 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).  相似文献   

6.
The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na+/P i cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state P i -induced currents had a voltage-independent apparent K m for P i of 0.03 mm and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mm Na+. The apparent K m for Na+ at 1 mm P i was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm at −30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage jumps elicited presteady-state relaxations in the presence of 96 mm Na+ which were suppressed at saturating P i (1 mm). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V 0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum P i -suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec−1. The voltage dependence of the relaxations was P i -independent, whereas changes in Na+ shifted V 0.5 to −60 mV at 25 mm Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement. Received: 11 March 1997/Revised: 3 June 1997  相似文献   

7.
Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl conductance (GCl), organic anion transport and Na+-dependent P i -uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and P i -induced currents and transport. NaPi-1 expression induced P i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P i (≥ 1 mm P i ). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl channel blocker NPPB, partially Na+-dependent and completely abolished in Cl free solution. In contrast, P i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl substitution. Endogenous P i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na+-dependent P i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P i -uptake system (K m for P i > 1 mm; partial Na+- and Cl-dependence; lack of NPPB block) were similar to the NaPi-1 induced P i -uptake, but no Ip could be recorded at P i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P i -uptake, but a P i -mediated modulation of GCl. Received: 22 October 1997/Revised: 24 March 1998  相似文献   

8.
In a previous report we documented an increased Na+-dependent transport of inorganic phosphate (P i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P i -uptake stimulator) lead to a 3-4-fold stimulation of Na+-dependent P i -uptake (10ng cRNA injected, 3–5 days of expression). Na+-independent uptake of P i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K m -values for the induced Na+-dependent uptake were 0.26 ± 0.04 mm for P i and 14.8 ± 3.0 mm for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P i -uptake into Xenopus laevis oocytes, but which is not a P i -transporter itself. Received: 31 July 1996/Revised: 16 October 1996  相似文献   

9.
K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li i ) but not external (Li o ) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K i of ∼7 mm. In Cl, Li i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li i reduced the V max and increased the K m for K efflux in Cl. Furthermore, Li i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume. Received: 1 November 1999/Revised: 6 June 2000  相似文献   

10.
Xenopus oocytes were injected with total RNA from chicory leaf tissues and then examined by the voltage-clamp technique. A double-step voltage protocol was used, with an initial hyperpolarization step from the holding potential of −35 to −140 mV followed by a second depolarization step to +60 mV. Two different outward currents were observed, one noninactivating (I ni ), and one inactivating (I i ). Only the noninactivating outward current (I ni ) could be induced by depolarization from −35 to +60 mV. The mean amplitude of I ni was 2915 ± 848 nA (n= 11). This current, carried by chloride ions, declined nearly to the baseline in 153 ± 64 sec (n= 13), and was highly dependent on intracellular calcium. After the rundown of I ni , the same oocyte was depolarized from −140 to +60 mV. This protocol induced an inactivating outward current (I i ) with a mean amplitude of 4461 ± 1605 nA (n= 13). I i was also carried by chloride ions and dependent on extracellular calcium. I i was strongly inhibited by 100 μm extracellular La3+. These two types of chloride currents were also observed after IP3 injection in control oocytes. I ni and I i were not observed in noninjected oocytes or water-injected oocytes. We suggest that the expression of total chicory leaf tissue RNA in Xenopus oocytes reveals a calcium homeostasis mechanism responsible for calcium mobilization from internal stores and subsequent calcium entry. Received: 22 May 1998/Revised: 2 October 1998  相似文献   

11.
The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999  相似文献   

12.
Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 × 10−4 and 2.7 × 10−5 nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10−2 and 10−4.  相似文献   

13.
The type IIa Na/Pi cotransporter mediates proximal tubular brush-border membrane secondary active phosphate (Pi) flux. It is rate limiting in tubular Pi reabsorption and, thus, a final target in many physiological and pathophysiological situations of altered renal Pi 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.  相似文献   

14.
In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for studying H+- and Na+-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (Pi) was accumulated by two kinetically discrete H+/Pi-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high Pi concentrations. The high-affinity system, subjected to regulation by both extracellular Pi availability and intracellular polyphosphate stores, is mobilized during Pi-starvation. In cells grown at pH 9.5–10, Pi uptake is mediated by several kinetically discrete Na+-dependent systems that are specifically activated by Na+ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high Pi concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during Pi-starvation and appear to be controlled by extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H+- and Na+-coupled Pi transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H+-coupled Pi transport systems are predominant. The contribution of the Na+/Pi cotransport systems to the total cellular Pi uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher. Received: 15 December 2000/Revised: 14 May 2001  相似文献   

15.
Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pH i ) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pH i in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH i decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pH i increase caused by elevating extracellular [K+], and (iii) an acid shift in the pH i dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pH i dependence of Na-H exchange, computed by adding the pH i dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment. Received: 14 October 1998/Revised: 2 March 1999  相似文献   

16.
In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl secretion. As recently proposed, beside its role of Cl channel, CFTR may regulate the activity of other channels such as a Ca2+-activated Cl channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca2+] i ([Ca2+] i ), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca2+] i in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca2+] i increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca2+] i in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca2+] i response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca2+] i by CFTR in CHO epithelial cell membranes. Received: 5 April 1999/Revised: 28 June 1999  相似文献   

17.
The type IIa Na+/Pi, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na+ and one divalent Pi 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 Pi transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of Pi 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 Pi-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 Pi 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.  相似文献   

18.
Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K+ and HCO 3 out of the cell, tending to lower intracellular pH (pHi). With an inwardly directed K/HCO3 gradient, the cotransporter mediates a net uptake of alkali (i.e., K+ and HCO 3 influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA+) inhibit the inwardly directed cotransporter by interacting at the intracellular K+ site. We computed the equivalent HCO 3 influx (J HCO3) mediated by the cotransporter from the rate of pHi increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pHi 8.0, using a dialysis fluid (DF) free of K+, Na+ and Cl. Our standard artificial seawater (ASW) also lacked Na+, K+ and Cl. After halting dialysis, we introduced an ASW containing 437 mm K+ and 0.5% CO2/12 mm HCO 3, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO2 influx, followed by a slower plateau-phase pHi increase, due to inward cotransport of K+ and HCO 3. With no QA+ in the DF, J HCO3 was ∼58 pmole cm−2 sec−1. With 400 mm tetraethylammonium (TEA+) in the DF, J HCO3 was virtually zero. The apparent K i for intracellular TEA+ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K+ channels. Introducing 100 mm inhibitor into the DF reduced J HCO3 to ∼20 pmole cm−2 sec−1 for tetramethylammonium (TMA+), ∼24 for TEA+, ∼10 for tetrapropylammonium (TPA+), and virtually zero for tetrabutylammonium (TBA+). The apparent K i value for TBA+ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA+), with an apparent K i of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO3 influx in the potency sequence PPTEA+ > TBA+ > TPA+ > TEA+≅ TMA+. The identification of inhibitors of the K/HCO3 cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter. Received: 21 November 2000/Revised: 14 May 2001  相似文献   

19.
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  相似文献   

20.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+] i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca2+] i in normal (16HBE14o cell line and primary lung culture) and in cystic fibrosis (CFTE29o cell line) human airway epithelia. The potency order of nucleotides on [Ca2+] i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+] i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o cells, the forskolin-induced [Ca2+] i response increased with increasing temperature. In CFTE29o cells, forskolin had no effect on [Ca2+] i at body temperature-forskolin-induced [Ca2+] i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+] i response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+] i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000  相似文献   

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