Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini

Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO(2)/HCO(3)(-) solution displayed all the hallmarks of an electrogenic Na(+)/HCO(3)(-) cotransporter: (a) a DIDS-sensitive pH(i) recovery following the initial CO(2)-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na(+)-dependent outward current under voltage-clamp conditions (-60 mV). All three variants had similar external HCO(3)(-) dependencies (apparent K(M) of 4-6 mM) and external Na(+) dependencies (apparent K(M) of 21-36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (-60 mV) expressing NBCe1-A exhibited peak HCO(3)(-)-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current-voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (V(m) = -60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO(3)(-)-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.     

Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase

Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 μM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.     

Carbonic anhydrase II increases the activity of the human electrogenic Na+/HCO3- cotransporter

Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a "transport metabolon." We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H(+) and Na(+) concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na(+) rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2-200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH(2)-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H(+) change following addition of CO(2)/HCO(3)(-) confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.     

A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1

Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na(+) before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9-19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11-21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by > or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.     

Binding of carbonic anhydrase IX to extracellular loop 4 of the NBCe1 Na+/HCO3- cotransporter enhances NBCe1-mediated HCO3- influx in the rat heart

Na(+)/HCO(3)(-) cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na(+):2 HCO(3)(-) into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO(3)(-) transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione-S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K(+) in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 μM) or 6-ethoxyzolamide (ETZ; 100 μM) (*P < 0.05). This depolarization protocol produced an intracellular pH (pH(i)) increase of 0.17 ± 0.01 (n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH(i) in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH(i) recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO(3)(-) influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.     

Functional characterization of human NBC4 as an electrogenic Na+-HCO cotransporter (NBCe2)

We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pH(i)) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO- and Na+-dependent currents. When we voltage clamped V(m) far from NBC4c's estimated reversal potential (E(rev)), the pH(i) recovery rate increased substantially. Both the currents and pH(i) recovery were blocked by 200 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimated the transporter's HCO:Na+ stoichiometry by measuring E(rev) at different extracellular Na+ concentration ([Na+]o) values. A plot of E(rev) against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute E(rev) values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.     

Inhibition of the Na/Bicarbonate Cotransporter NBCe1-A by diBAC Oxonol Dyes Relative to Niflumic Acid and a Stilbene

Na/HCO(3) cotransporters (NBCs) are important regulators of intracellular pH (pH(i) in a variety of organ systems where acid-base status is critical for tissue function. To characterize the pharmacology of NBCs in more detail, we used the two-electrode voltage-clamp technique to examine the effect of previously identified inhibitors of anion exchanger 1 (AE1) on the activity of rat NBCe1-A expressed in Xenopus laevis oocytes. NBC-expressing oocytes voltage-clamped at -60 mV and exposed to a 5% CO(2)/33 mM HCO(3)(-) solution displayed NBC-mediated outward currents that were inhibited by either niflumic acid or one of the two bis-oxonol dyes diBA(3)C4 and diBA(5)C4. NBCe1-A was less sensitive to niflumic acid (apparent K(i) of 100 microM) than 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, apparent K(i) of 36 microM) but more sensitive to the diBAC dyes (apparent K(i) of approximately 10 microM). Based on current-voltage relationships, the diBAC dyes inhibited HCO(3)(-) -induced NBCe1-mediated inward currents more so than outward currents. NBCe1 sensitivity to the dyes was (1) lower in the presence of 40 microM DIDS, (2) unaffected by changes in external HCO(3)(-) concentration and (3) only modestly higher at an external Na(+) concentration of 5, but not 15 or 33, mM. Therefore, the diBAC dyes compete with DIDS but not appreciably with Na(+) or HCO(3)(-) for binding. The mechanism of diBAC inhibition of NBCe1 appears similar to that previously reported for AE1.     

Voltage dependence of H+ buffering mediated by sodium bicarbonate cotransport expressed in Xenopus oocytes

The electrogenic sodium bicarbonate cotransporter (NBCe1) is expressed in many epithelial cells and, in the brain, in glial cells. Little is known about the physiological significance of the NBCe1 for proton homeostasis and for other acid/base-coupled transporters in these cells. We have measured the voltage-dependent transport activity of an NBC from human kidney, type hkNBCe1, expressed in oocytes of the frog Xenopus laevis, by recording membrane current and the changes in intracellular pH and sodium at different membrane potentials between -20 and -100 mV. The apparent intracellular buffer capacity was increased and became dependent upon membrane voltage when the NBCe1 was expressed; the measured buffer capacity increased by up to 7 mm/10 mV of membrane depolarization. Lactate transport by the electroneutral monocarboxylate transporter became enhanced and dependent upon membrane potential, when the monocarboxylate transporter (isoform 1) was co-expressed with NBCe1 in oocytes. Our results indicate that the electrogenic NBCe1 renders the cell membrane potential an effective regulator of intracellular H(+) buffering and acid/base-coupled metabolite transport.     

Cloning and characterization of an electrogenic Na/HCO3- cotransporter from the squid giant fiber lobe

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na(+)-driven Cl-HCO(3)(-) exchanger, sqNDCBE--related to the SLC4 superfamily and cloned from giant fiber lobe cDNA--is the only HCO(3)(-)-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5' and 3' directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na(+)-coupled SLC4 transporters. However, when we measure pH(i) and membrane potential--or use two-electrode voltage clamping to measure currents--on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO(3)(-) cotransporter, NBCe. That is, exposure to extracellular CO(2)/HCO(3)(-) not only causes a fall in pH(i), followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pH(i) recovery and current require HCO(3)(-) and Na(+), and are blocked by DIDS. Furthermore, neither K(+) nor Li(+) can fully replace Na(+) in supporting the pH(i) recovery. Extracellular Cl(-) is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.     

Enhanced activity of ventricular Na+-HCO3- cotransport in pressure overload hypertrophy

The Na(+)-HCO(3)(-) cotransporter (NBC) plays a key role in intracellular pH (pH(i)) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pH(i) was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC (J(NBC)) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na(+)/H(+) exchange was also increased in hypertrophied myocytes, the relative enhancement of J(NBC) was larger, 6) membrane depolarization markedly increased J(NBC) in hypertrophied myocytes, and 7) losartan, an ANG II AT(1) receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na(+) overload, which may render the ventricles more vulnerable to Ca(2+) overload during ischemia-reperfusion.     

The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia

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     

Measurement of pH to quantify urease activity.

The rate of change of pH caused by the hydrolysis of urea was measured for urease solutions of 18 different concentrations. Concentrations were converted into an activity, A (measured in IU/cm3), by using a titrimetric method to assay the urease sample. For activities in the range 0.6-38 IU/cm3, A was related to the initial rate of change of pH, (dpH/dt)0, (measured in s-1) by the empirical relationship: A = 549(dpH/dt)0-1423(dpH/dt)2(0). Values of (dpH/dt)0 were sensitive to changes in the urease activity of about 0.6 IU/cm3.     

Evidence against a direct interaction between intracellular carbonic anhydrase II and pure C-terminal domains of SLC4 bicarbonate transporters

Based on solid-phase binding assays with enzyme-linked immunosorbent assay detection, previous investigators suggested that intracellular carbonic anhydrase II (CA II) interacts at high affinity with the C-terminal (Ct) domains of SLC4 bicarbonate-transport proteins, expressed as glutathione S-transferase (GST) fusion proteins, to form functional HCO3- metabolons. Here we re-evaluated this protein-protein interaction using two solid-phase binding assays. We first compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1), and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. We found that when expressed as GST fusion proteins, all three bind to CA II (Kd 300-600 nM) better than does pure GST. However, we detected no binding of pure SLC4-Ct peptides to immobilized CA II. Second, we reversed assay orientation by immobilizing the SLC4-Ct fusion proteins or peptides. We found that more CA II binds to GST than to any of the three GST-SLC4-Ct fusion proteins. Furthermore, we detected no binding of CA II to any of the immobilized pure SLC4-Ct peptides. Finally, we used surface plasmon resonance to detect possible rapid interactions between CA II and the pure peptides. Although we detected acetazolamide binding to immobilized CA II and specific antibodies binding to immobilized SLC4-Ct peptides, we detected no binding of CA II to immobilized SLC4-Ct or vice versa. Thus, although an HCO3 metabolon may exist, CA II cannot bind directly to pure SLC4-Ct peptides and can bind to GST-SLC4-Ct fusion proteins only when the CA II is immobilized and the fusion protein is soluble, and not vice versa.     

Direct extracellular interaction between carbonic anhydrase IV and the human NBC1 sodium/bicarbonate co-transporter

Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pH(i)) and HCO(3)(-) metabolism. Electrogenic NBC1 catalyzes HCO(3)(-) fluxes in mammalian kidney, pancreas, and heart cells. Carbonic anhydrase IV (CAIV), which is also present in these tissues, is glycosylphosphatidyl inositol-anchored to the outer surface of the plasma membrane where it catalyzes the hydration-dehydration of CO(2)/HCO(3)(-). The physical and functional interactions of CAIV and NBC1 were investigated. NBC1 activity was measured by changes of pH(i) in NBC1-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of pH(i) recovery by 44 +/- 3%, as compared to NBC1-alone. In contrast, CAIV did not increase the functional activity of G767T-NBC1 (mutated on the fourth extracellular loop (EC4) of NBC1), and G767T-NBC1, unlike wild-type NBC1, did not interact with CAIV in glutathione-S-transferase pull-down assays. This indicates that G767 of NBC1 is directly involved in CAIV interaction. NBC1-mediated pH(i) recovery rate after acid load was inhibited by 40 +/- 7% when coexpressed with the inactive human CAII mutant, V143Y. V143Y CAII competes with endogenous CAII for interaction with NBC1 at the inner surface of the plasma membrane, which indicates that NBC1/CAII interaction is needed for full pH(i) recovery activity. We conclude that CAIV binds EC4 of NBC1, and this interaction is essential for full NBC1 activity. The tethering of CAII and CAIV close to the NBC1 HCO(3)(-) transport site maximizes the transmembrane HCO(3)(-) gradient local to NBC1 and thereby activates the transport rate.     

Plasmalemmal H+ extruders in mammalian alveolar macrophages

The distribution of plasmalemmal V-type H+-pumps (V-ATPase) among mammalian macrophages (mvarphi) is uncertain and, hence, the functional significance of mvarphi plasmalemmal V-ATPase is unclear. This study investigated the role of V-ATPase in the regulation of intracellular pH (pH(i)) by resident alveolar mvarphi from sheep, pigs, dogs and rabbits. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pH(i) and the rate of pH(i) recovery (dpH(i)/dt) from intracellular acid-loads (NH(4)Cl prepulse). Baseline pH(i) was 7.1-7.2. In sheep, pig and dog studies, 10 microM bafilomycin A(1) (a selective V-ATPase inhibitor) caused a rapid fall in baseline pH(i) (0.15-0.20 units); baseline values were unaffected by 0.1 mM amiloride (a Na+ transport inhibitor). V-ATPase activity (bafilomycin-sensitive component of dpH(i)/dt) was solely responsible for pH(i) recovery from intracellular acid-loads at acid-loaded pH(i) values >6.8-6.9. Na+/H+ exchange (amiloride-sensitive component of dpH(i)/dt) was detected only at acid-loaded pH(i) values <6.8. The activity of both H+ extruders increased at lower pH(i) values, albeit the Na+/H+ exchanger was more pH-sensitive than was V-ATPase. In rabbit studies, 10 microM bafilomycin A(1) and 1 mM N-ethylmaleimide (a non-specific H+-pump inhibitor) produced similar falls in baseline mvarphi pH(i), but had significantly larger effects than did the selective V-ATPase inhibitor concanamycin A (相似文献   

Oligomeric structure and minimal functional unit of the electrogenic sodium bicarbonate cotransporter NBCe1-A

The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wild-type (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys(630) and Cys(642) in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys(992) construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-A(T442C) mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.     

The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na⁺ and HCO₃^? into the cytoplasm upon intracellular acidosis, restoring the acidic pH_i to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na⁺:1HCO₃^?) and the electrogenic NBCe1 (1Na⁺:2HCO₃^?). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.     

Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+-HCO3− cotransporter NBCe1 expressed in Xenopus oocytes

The electrogenic Na⁺-HCO₃^? cotransporter NBCe1 plays essential roles in the regulation of systemic and/or local pH. Homozygous inactivating mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. We recently showed that defective membrane expression of NBCe1, caused by several mutations such as Delta65bp (S982NfsX4), is also associated with familial migraine. The Delta65bp mutant is quite unique in that it lacks a putative carbonic anhydrase (CA) II-binding domain but still shows an apparently normal transport activity in Xenopus oocytes. In this addendum, we show that the co-expression of CAII together with the wild-type NBCe1 or the Delta65bp mutant does not enhance the NBCe1 activities in oocytes. Moreover, a carbonic anhydrase inhibitor acetazolamide fails to inhibit the wild-type or the Delta65bp activities co-expressed with CAII. These results indicate that a bicarbonate transport metabolon proposed for the interaction between CAII and NBCe1 does not work at least in Xenopus oocytes.     

Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+ -HCO₃- cotransporter NBCe1 expressed in Xenopus oocytes

The electrogenic Na+ -HCO?? cotransporter NBCe1 plays essential roles in the regulation of systemic and/or local pH. Homozygous inactivating mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. We recently showed that defective membrane expression of NBCe1, caused by several mutations such as Delta65bp (S982NfsX4), is also associated with familial migraine. The Delta65bp mutant is quite unique in that it lacks a putative carbonic anhydrase (CA) II-binding domain but still shows an apparently normal transport activity in Xenopus oocytes. In this addendum, we show that the co-expression of CAII together with the wild-type NBCe1 or the Delta65bp mutant does not enhance the NBCe1 activities in oocytes. Moreover, a carbonic anhydrase inhibitor acetazolamide fails to inhibit the wild-type or the Delta65bp activities co-expressed with CAII. These results indicate that a bicarbonate transport metabolon proposed for the interaction between CAII and NBCe1 does not work at least in Xenopus oocytes.