首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Intestinal absorption offolates has been characterized as a facilitative process with a low pHoptimum. Studies with intestinal epithelial cells have suggested thatthis activity is mediated by the reduced folate carrier (RFC1). In thispaper, we report on folate transport characteristics in an immortalizedrat IEC-6 cell line that was found to exhibit the predominant influxactivity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs andhigh folic acid uptake only at the low pH. For the two clones with thehighest level of transport activity, relative MTX influx at the two pHswas reversed. Moreover, the low pH MTX influx activity([MTX]e = 0.5 µM) was markedly inhibited by 20 µM folic acid while influx at neutral pH was not. Furthermore, in thepresence and absence of glucose at low pH, MTX and folic acid influxactivity was inhibited by azide, while MTX influx at pH 7.4 wasstimulated by azide in the absence of glucose but was unchanged in thepresence of glucose and azide. This was contrasted with the results oftransfection of the same RFC1 construct into an L1210 murine leukemiacell line bearing a nonfunctional endogenous carrier. In this case, theactivity expressed was only at pH 7.4. These data indicate that RFC1can exhibit two distinct types of folate transport activities inintestinal cells that must depend on tissue-specific modulators.

  相似文献   

2.
Regulation of the epithelial Na(+) channel by extracellular acidification   总被引:2,自引:0,他引:2  
The effect of extracellular acidification wastested on the native epithelial Na+ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pHo) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (INa)by 68.4 ± 11% (n = 9) at 60 min. This increaseof INa was attributed to an increase of openchannel and total channel (NT) densities. Similar changes were observed with pHo 5.4. The effects ofpHo were blocked by buffering intracellularCa2+ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pHo 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pHostimulate the native ENaC by increasing NT butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

  相似文献   

3.
Dynamics of calcium regulation of chloride currents in Xenopus oocytes   总被引:1,自引:0,他引:1  
Ca-activated Cl currents are widely expressed in many cell typesand play diverse and important physiological roles. TheXenopus oocyte is a good model systemfor studying the regulation of these currents. We previously showedthat inositol 1,4,5-trisphosphate (IP3) injection intoXenopus oocytes rapidly elicits anoninactivating outward Cl current(ICl1-S)followed several minutes later by the development of slow inward(ICl2) andtransient outward(ICl1-T) Clcurrents. In this paper, we investigate whether these three currentsare mediated by the same or different Cl channels. Outward Cl currentswere more sensitive to Ca than inward Cl currents, as shown byinjection of different amounts of Ca or by Ca influx through aheterologously expressed ligand-gated Ca channel, the ionotropicglutamate receptor iGluR3. These data could be explained by twochannels with different Ca affinities or one channel with a higher Caaffinity at depolarized potentials. To distinguish between thesepossibilities, we determined the anion selectivity of the threecurrents. The anion selectivity sequences for the three currents werethe same (I > Br > Cl), butICl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, afterconsideration of possible errors, the hypothesis that a single type ofCl channel underlies the complex waveforms of the three differentmacroscopic Ca-activated Cl currents inXenopus oocytes remains a viable alternative.

  相似文献   

4.
Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln420. A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser135 was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC. folic acid; folate absorption; methotrexate  相似文献   

5.
Role of reduced folate carrier in intestinal folate uptake   总被引:3,自引:0,他引:3  
Studies from our laboratory and others have characterized different aspects of the intestinal folate uptake process and have shown that the reduced folate carrier (RFC) is expressed in the gut and plays a role in the uptake process. Little, however, is known about the actual contribution of the RFC system toward total folate uptake by the enterocytes. Addressing this issue in RFC knockout mice is not possible due to the embryonic lethality of the model. In this study, we describe the use of the new approach of lentivirus-mediated short hairpin RNA (shRNA) to selectively silence the endogenous RFC of the rat-derived intestinal epithelial cells (IEC-6), an established in vitro model for folate uptake, and examined the effect of such silencing on folate uptake. First we confirmed that the initial rate of [(3)H]folic acid uptake by IEC-6 cells was pH dependent with a markedly higher uptake at acidic compared with alkaline pH. We also showed that the addition of unlabeled folic acid to the incubation buffer leads to a severe inhibition ( approximately 95%) in [(3)H]folic acid (16 nM) uptake at buffer pH 5.5 but not at buffer pH 7.4. We then examined the effect of treating (for 72 h) IEC-6 cells with RFC-specific shRNA on the levels of RFC protein and mRNA and observed substantial reduction in the levels of both parameters ( approximately 80 and 78%, respectively). Such a treatment was also found to lead to a severe inhibition ( approximately 90%) in initial rate of folate uptake at buffer pH 5.5 (but not at pH 7.4); uptake of the unrelated vitamin, biotin, on the other hand, was not affected by such a treatment. These results demonstrate that the RFC system is the major (if not the only) folate uptake system that is functional in intestinal epithelial cells.  相似文献   

6.
Members of the SLC20 family or type III Na+-coupled Pi cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular Pi homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic Pi cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na+-dependent 32Pi uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li+ substituted for Na+. The dependence of the Pi-induced current on Pi concentration was Michaelian, and the dependence on Na+ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent Pi affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual 22Na-32Pi uptake and suggested a 2:1 Na+:Pi stoichiometry. A correlation of 32Pi uptake and net charge movement indicated one charge translocation per Pi. Changes in oocyte surface pH were consistent with transport of monovalent Pi. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na+:H2PO4:Na+. Significantly, in contrast to type II Na+-Pi cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na+-Pi cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor  相似文献   

7.
A recent study onXenopus oocytes [N. L. Nakhoul,M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (CellPhysiol. 43): C543-548, 1998] injected withcarbonic anhydrase showed that expressing aquaporin 1 (AQP1) increasesby ~40% the rate at which exposing the cell toCO2 causes intracellular pH tofall. This observation is consistent with several interpretations.Overexpressing AQP1 might increase apparentCO2 permeability by1) allowingCO2 to pass through AQP1,2) stimulating injected carbonicanhydrase, 3) enhancing theCO2 solubility of the membrane'slipid, or 4) increasing theexpression of a native "gas channel." The purpose of the presentstudy was to distinguish among these possibilities. We found thatexpressing the H2O channel AQP1 inXenopus oocytes increases theCO2 permeability of oocytes in anexpression-dependent fashion, whereas expressing theK+ channel ROMK1 has no effect.The mercury derivativep-chloromercuriphenylsulfonic acid(PCMBS), which inhibits the H2Omovement through AQP1, also blocks the AQP1-dependent increase inCO2 permeability. Themercury-insensitive C189S mutant of AQP1 increases theCO2 permeability of the oocyte tothe same extent as does the wild-type channel. However, the C189S-dependent increase in CO2permeability is unaffected by treatment with PCMBS. These data rule outoptions 2-4 listed above. Thusour results suggest that CO2passes through the pore of AQP1 and are the first data to demonstratethat a gas can enter a cell by a means other than diffusing through themembrane lipid.

  相似文献   

8.
Intestinal and renalabsorption of inorganic phosphate (Pi) is critical forphosphate homeostasis in mammals. We have isolated a cDNA that encodesa type III Na-dependent phosphate cotransporter from mouse smallintestine (mPit-2). The nucleotide sequence of mPit-2 predicts aprotein of 653 amino acids with at least 10 putative transmembranedomains. Kinetic studies, carried out in Xenopus oocytes,showed that mPit-2 cRNA induces significant Na-dependent Piuptake with an apparent Michaelis constant (Km)for phosphate of 38 µM. The transport of phosphate by mPit-2 isinhibited at high pH. Northern blot analysis demonstrated the presenceof mPit-2 mRNA in various tissues, including intestine, kidney, heart,liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed thatmPit-2 mRNA is expressed throughout the vertical crypt-villus axis ofthe intestinal epithelium. The presence of mPit-2 in the mouseintestine and its unique transport characteristics suggest thatmultiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.

  相似文献   

9.
It is generally accepted that gases such asCO2 cross cell membranes bydissolving in the membrane lipid. No role for channels or pores in gastransport has ever been demonstrated. Here we ask whether expression ofthe water channel aquaporin-1 (AQP1) enhances theCO2 permeability ofXenopus oocytes. We expressed AQP1 inXenopus oocytes by injecting AQP1cRNA, and we assessed CO2permeability by using microelectrodes to monitor the changes inintracellular pH (pHi) producedby adding 1.5% CO2/10 mM to (or removing it from) theextracellular solution. Oocytes normally have an undetectably low levelof carbonic anhydrase (CA), which eliminates theCO2 hydration reaction as arate-limiting step. We found that expressing AQP1 (vs. injectingwater) had no measurable effect on the rate ofCO2-inducedpHi changes in such low-CAoocytes: adding CO2 causedpHi to fall at a mean initial rateof 11.3 × 104 pHunits/s in control oocytes and 13.3 × 104 pH units/s in oocytesexpressing AQP1. When we injected oocytes with water, and a few dayslater with CA, the CO2-inducedpHi changes in these water/CAoocytes were more than fourfold faster than in water-injected oocytes(acidification rate, 53 × 104 pH units/s).Ethoxzolamide (ETX; 10 µM), a membrane-permeant CA inhibitor, greatlyslowed the pHi changes (16.5 × 104 pHunits/s). When we injected oocytes with AQP1 cRNA and then CA, theCO2-inducedpHi changes in these AQP1/CAoocytes were ~40% faster than in the water/CA oocytes (75 × 104 pH units/s), and ETXreduced the rates substantially (14.7 × 104 pH units/s). Thus, inthe presence of CA, AQP1 expression significantly increases theCO2 permeability of oocytemembranes. Possible explanations include1) AQP1 expression alters the lipidcomposition of the cell membrane, 2)AQP1 expression causes overexpression of a native gas channel,and/or 3) AQP1 acts as achannel through which CO2 canpermeate. Even if AQP1 should mediate aCO2 flux, it would remain to bedetermined whether this CO2movement is quantitatively important.

  相似文献   

10.
The role ofintracellular pH (pHi) in regulation of AE2 function inXenopus oocytes remains unclear. We therefore compared AE2-mediated 36Cl efflux fromXenopus oocytes during imposed variation of extracellular pH(pHo) or variation of pHi at constantpHo. Wild-type AE2-mediated 36Clefflux displayed a steep pHo vs. activity curve, withpHo(50) = 6.91 ± 0.04. SequentialNH2-terminal deletion of amino acid residues in tworegions, between amino acids 328 and 347 or between amino acids 391 and510, shifted pHo(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocytepHi at constant pHo and were shown to beneither substrates nor inhibitors of AE2-mediated Cltransport. At constant pHo, AE2 was inhibited byintracellular acidification and activated by intracellularalkalinization. Our data define structure-function relationships withinthe AE2 NH2-terminal cytoplasmic domain, which demonstratesdistinct structural requirements for AE2 regulation by intracellularand extracellular protons.

  相似文献   

11.
After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) Na+ independent; 4) saturable as a function of substrate concentration (apparent Km = 0.762 ± 0.10 µM); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation  相似文献   

12.
Cl- currents activated via purinergic receptors in Xenopus follicles   总被引:2,自引:0,他引:2  
Ionic currents elicited via purinergic receptors located in themembrane of Xenopus follicles werestudied using electrophysiological techniques. Follicles responded toATP-activating inward currents with a fast time course(Fin). InRinger solution, reversal potential (Erev) ofFin was 22mV, which did not change with external substitutions ofNa+ orK+, whereas solutions containing50 or 5% of normal Clconcentration shiftedErev to about +4and +60 mV, respectively, and decreasedFin amplitude,indicating thatFin was carriedby Cl.Fin had an onsetdelay of ~400 ms, measured by application of a brief jet of ATP froma micropipette positioned near the follicle (50 µm).Fin was inhibitedby 50% in follicles pretreated with pertussis toxin. This suggests a Gprotein-mediated receptor channel pathway.Fin was mimickedby 2-MeSATP and UTP, the potency order (half-maximal effectiveconcentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP(1,086 nM). All agonists generatedCl currents and displayedcross-inhibition on the others.Fin activation byacetylcholine also cross-inhibitedFin-ATPresponses, suggesting that all act on a common channel-activationpathway.

  相似文献   

13.
The human electrogenic renal Na-HCO3 cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO3 from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na+ affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. bicarbonate; intracellular pH; acidbase; SLC4A4; Na+-HCO3 cotransporter 1  相似文献   

14.
We have clonedand functionally characterized the human Na+-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa+ [concentration of substrate necessary for half-maximaltransport (Kt) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa+-to-succinate stoichiometry is 3:1 and concentration ofNa+ necessary for half-maximal transport(KNa+0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na+-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,KSuc0.5 is 102 ± 20 µM andKNa+0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na+-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li+inhibits succinate-induced currents in the presence of Na+.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns.

  相似文献   

15.
Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pHi) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl/Cl and Cl/HCO3 exchange are inhibited by acid pHi or extracellular pH (pHo). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl/Cl exchange, measured as DIDS-sensitive 36Cl efflux from Xenopus oocytes, was experimentally altered by varying pHi at constant pHo or varying pHo. Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal 36Cl efflux at pHo 7.4 and acid shifted the pHo vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl transport activity with retention of pHo sensitivity. In contrast to the effects of DEPC on wild-type AE2, pHo sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pHi, pHi sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pHi and pHo. pH regulation; histidine residues; Cl/HCO3 exchange  相似文献   

16.
The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidneyHEK-293 cells, and the transport characteristics were measured in cellstransfected with a vector containing NaPi-3 or with the vector alone(sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na andPO4 with K Na1/2 of 128 mM(PO4 = 0.1 mM) andK PO41/2of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfectedcells expressing the transporter. Transfection had no effect on theNa-independent 32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenateinhibited flux with an inhibition constant of 0.4 mM. The phosphatetransport in sham- and NaPi-3-transfected cells has nearly the sametemperature dependence in the absence and presence of extracellularNa. The Na-dependent phosphate flux decreased with pH insham-transfected cells but was pH independent in transfected cells. TheNa-dependent32PO4influx was inhibited byp-chloromercuriphenylsulfonate,phosphonoformate, phloretin, vanadate, and5-(N-methyl-N-isobutyl)-amiloridebut not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior ofNaPi-3 homologues in the renal tubule of other species and, thus,demonstrate the fidelity of this transfection system for the study ofthis protein. Commensurate with the increased functional expression,there was an increase in the amount of NaPi-3 protein by Westernanalysis.

  相似文献   

17.
The slc4 and slc26 gene families encode two distinct groups of gene products that transport HCO3 and other anions in mammalian cells. The SLC4 and SLC26 proteins are important contributors to transepithelial movement of fluids and electrolytes and to cellular pH and volume regulation. Herein we describe the cDNA cloning from the nematode Caenorhabditis elegans of four anion bicarbonate transporter (abts) homologs of slc4 cDNA and eight sulfate permease (sulp) homologs of slc26 cDNA. Analysis of transgenic nematode strains carrying promoter::GFP fusions suggests relatively restricted expression patterns for many of these genes. At least three genes are expressed primarily in the intestine, three are expressed primarily in the excretory cell, and one is expressed in both of these polarized cell types. One of the genes is also expressed exclusively in the myoepithelium-like cells of the pharynx. Many of the sulp gene products localize to the basolateral membrane rather than to the apical membrane. Several ABTS and SULP proteins exhibited anion transport function in Xenopus oocytes. The strongest Cl transporter among these also mediated Cl/HCO3 exchange. These findings encourage exploitation of the genetic strengths of the nematode model system in the study of the physiological roles of anion transport by the proteins of these two highly conserved gene families.  相似文献   

18.
Thecharacteristics of L-lactic acid transport across thetrophoblast basal membrane were investigated and compared with those across the brush-border membrane by using membrane vesicles isolated from human placenta. The uptake ofL-[14C]lactic acid into basal membranevesicles was Na+ independent, and an uphill transport wasobserved in the presence of a pH gradient([H+]out > [H+]in).L-[14C]lactic acid uptake exhibitedsaturation kinetics with a Km value of 5.89 ± 0.68 mM in the presence of a pH gradient.p-Chloromercuribenzenesulfonate and-cyano-4-hydroxycinnamate inhibited the initial uptake, whereas phloretin or 4,4'-diisothiocyanostilbene-2,2'-disulfonate did not.Mono- and dicarboxylic acids suppressed the initial uptake. Inconclusion, L-lactic acid transport in the basal membraneis H+ dependent and Na+ independent, as is alsothe case for the brush-border membrane transport, and itscharacteristics resemble those of monocarboxylic acid transporters.However, there were several differences in the effects of inhibitorsbetween basal and brush-border membrane vesicles, suggesting that thetransporter(s) involved in L-lactic acid transport in thebasal membrane of placental trophoblast may differ from those in thebrush-border membrane.

  相似文献   

19.
TheShakerBK+ channel was used as a modelvoltage-gated channel to probe the interaction of volatile generalanesthetics with gating mechanisms. The effects of three anesthetics,chloroform (CHCl3), isoflurane,and halothane, were studied using recombinant native and mutantShaker channels expressed inXenopus oocytes. Gating currents andmacroscopic ionic currents were recorded with the cut-open oocytevoltage-clamp technique. The effects ofCHCl3 and isoflurane on gatingkinetics of noninactivating mutants were opposite, whereas halothanehad no effect. The effects on ionic currents were also agent dependent:CHCl3 and halothane produced areduction of the macroscopic conductance, whereas isoflurane increasedit. The results indicate that the gating machinery of the channel ismostly insensitive to the anesthetics during activation until near theopen state. The effects on the conductance are mainly due to changes inthe transitions in and out of the open state. The data give support todirect protein-anesthetic interactions. The magnitude and nature of theeffects invite reconsideration ofShaker-likeK+ channels as important sites ofaction of general anesthetics.

  相似文献   

20.
The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca2+-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca2+ concentration([Ca2+]i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa2+-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca2+. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca2+ displacement of intracellularH+ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO2 or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca2+uptake, resulting in inhibition of cytosolicH+ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca2+-ATPase(i.e.,Ca2+/H+exchanger), including La3+,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca2+-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca2+]i increase.

  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号