Role Of HCO3 Research Articles

Potent and selective inhibition of anion exchange activity of SLC26A9 by A9‐301

SLC26A9 (solute carrier family 26, member 9) is highly expressed epithelial cells of the respiratory tract, stomach, pancreas and kidney, and acts as a Cl‐/HCO3‐ exchanger. SLC26A9 plays a pivotal role in HCO3‐ secretion in lung, and emerging evidence suggests that SLC26A9 is a therapeutic target for cystic fibrosis (CF) and potentially other mucosal obstructive pulmonary diseases. In this study, a cell‐based high‐throughput screening (HTS) assay was established and performed to identify highly potent and selective small molecule inhibitors of SLC26A9. Screening of 30,000 synthetic small molecules identified three novel SLC26A9 inhibitors. Unlike the previous non‐selective SLC26A9 inhibitors, the novel SLC26A9 inhibitors potently and selectively inhibited anion exchange activity of SLC26A9. The most potent inhibitor, A9‐301, significantly blocked Cl‐/I‐ exchange activity of SLC26A9 (IC50 of ~100 nM) without affecting other anion exchanges and chloride channels, including SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A7, CFTR and ANO1. These results suggest that the novel potent and selective inhibitors of SLC26A9 are useful tools for pharmacological dissection of SLC26A9 and may be potential therapeutic candidates for airway inflammatory diseases.

The role of HCO3\u2013 in propionate-induced anion secretion across rat caecal epithelium

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl− secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-induced Isc (IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl−- or nominally HCO3−-free buffer. In the case of Cl−, the dependency was restricted to the presence of Cl− in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl−-cotransporter, only numerically reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl−. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3−-cotransporters in caecum, which might underlie the observed Na+ dependency. These results suggest that propionate sensing in caecum is coupled to HCO3– secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.

Structure and function of LCI1: a plasma membrane CO2 channel in the Chlamydomonas CO2 concentrating mechanism.

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO2 in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO2 concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (Ci ) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active Ci uptake systems. Fundamental to eukaryotic Ci uptake systems are Ci transporters/channels located in membranes of various cell compartments, which together facilitate the movement of Ci from the environment into the chloroplast, where primary CO2 assimilation occurs. Two putative plasma membrane Ci transporters, HLA3 and LCI1, are reportedly involved in active Ci uptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO3- transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full-length LCI1 membrane protein to reveal LCI1 structural characteristics, as well as in vivo physiological studies in an LCI1 loss-of-function mutant to reveal the Ci species preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO2 uptake and that LCI1 likely functions as a plasma membrane CO2 channel, possibly a gated channel.

Tunable Ag Micromorphologies Show High Activities for Electrochemical H2 Evolution and CO2 Electrochemical Reduction

Contaminate-free nanostructured Ag is prepared from electrochemical redox-treated Ag by two different potential pulses (DP and r-DP) in an NaBr aqueous solution. Agr-DP and AgDP from r-DP and DP, respectively, are easily tuned for their activities in the hydrogen evolution reaction (HER) favoring or selective CO2 reduction reaction (CO2RR). Agr-DP shows Pt-like HER activity in H2SO4 aqueous solution, and AgDP is capable of electrochemically reducing CO2 to CO with approximately 97.8% catalytic selectivity at a low overpotential of ∼0.25 V in NaHCO3 aqueous solution. The electrochemical signals for the first observed CO2•– intermediate adsorption (CO2ads•–) and HCO3– adsorption (HCO3–ads) in the selected NaClO4 aqueous solution were used to understand the possible role of HCO3– in CO2RR.

Role of HCO3- and Cl- in the Pitting of 20CrMo Steel in simulated oil field environment

Role of HCO3- and Cl- in the Pitting of 20CrMo Steel in simulated oil field environment

Stomatin modulates the activity of the Anion Exchanger 1 (AE1, SLC4A1)

Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3− and Cl− exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl−/HCO3− exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3− and outward Cl− gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3− for stomatin-deficient patients. Similarly, kinetics of Cl− efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.

Solar photocatalytic degradation of bisphenol A with CuOx/BiVO4: Insights into the unexpectedly favorable effect of bicarbonates

A series of CuOx/BiVO4 catalysts of variable copper loading (0–3.0wt.% Cu) were synthesized and characterized with the use of several techniques, including X-ray diffraction (XRD), scanning electron microscopy (SEM–EDS), high resolution transmission electron microscopy (HR-TEM) and UV–vis diffuse reflectance spectroscopy (DRS). Results show that all samples exhibit the monoclinic scheelite structure of BiVO4 and have similar band gaps (2.25–2.30eV) and specific surface areas (0.6–1.5m2/g). Copper is well-dispersed on the surface of BiVO4 in the form of small (<5nm) CuOx (most possibly CuO) crystallites. The photocatalytic activity of the synthesized materials was evaluated for the degradation of bisphenol A (BPA) under artificial visible- and solar-light irradiation. Pure BiVO4 was found to be active for the title reaction and its photocatalytic performance is substantially improved following addition of copper. Optimal results were obtained for the catalyst containing 0.75wt.% Cu, which was further investigated to study the effects of operating conditions and water matrix on catalytic performance. Results show that the rate of BPA degradation increases dramatically (by a factor of 15) in the presence of bicarbonate (HCO3−), compared to ultrapure water. The role of HCO3− was investigated with EPR and DMPO- and PBN-spin-trapping experiments, which allowed quantification of hydroxyl (OH) and carbonate (CO3−) radicals. It was shown that significant amounts of OH and CO3− radicals are photo-produced over both the pure and the copper-loaded BiVO4, with the rate of radicals formation being 3 times higher for the latter catalyst. Results are explained considering the efficient separation of photogenerated electron-hole pairs at the p–n heterojunction developed between the n-type BiVO4 and the p-type CuO (or Cu2O) semiconductors. In contrast to the OH radicals, which diffuse away from the particle surface, the CO3− radicals accumulate at the particle interface. The by-products of BPA degradation were identified by LC–MS–TOF over the Cu-loaded catalyst and results were used to propose possible reaction pathways. It is concluded that the exceptionally high photodegradation rate observed in the presence of HCO3− can be attributed to the formation of large amounts of OH radicals in the bulk solution, which react with BPA to yield reaction by-products through hydroxylation and scission of the bond between the isopropylidene carbon and the phenyl group.

Potassium channels in pancreatic duct epithelial cells: their role, function and pathophysiological relevance.

Pancreatic ductal epithelial cells play a fundamental role in HCO3 (-) secretion, a process which is essential for maintaining the integrity of the pancreas. Although several studies have implicated impaired HCO3 (-) and fluid secretion as a triggering factor in the development of pancreatitis, the mechanism and regulation of HCO3 (-) secretion is still not completely understood. To date, most studies on the ion transporters that orchestrate ductal HCO3 (-) secretion have focussed on the role of Cl(-)/HCO3 (-) exchangers and Cl(-) channels, whereas much less is known about the role of K(+) channels. However, there is growing evidence that many types of K(+) channels are present in ductal cells where they have an essential role in establishing and maintaining the electrochemical driving force for anion secretion. For this reason, strategies that increase K(+) channel function may help to restore impaired HCO3 (-) and fluid secretion, such as in pancreatitis, and therefore provide novel directions for future pancreatic therapy. In this review, our aims are to summarize the types of K(+) channels found in pancreatic ductal cells and to discuss their individual roles in ductal HCO3 (-) secretion. We will also describe how K(+) channels are involved in pathophysiological conditions and discuss how they could act as new molecular targets for the development of therapeutic approaches to treat pancreatic diseases.

Selective oxidation over a metalloporphyrinic metal–organic framework catalyst and insights into the mechanism of bicarbonate ion as co-catalyst

A biomimetic oxidation catalyst, metalloporphyrinic metal–organic framework, was successfully constructed by manganese tetrakis (4-carboxyphenyl) porphyrin as a bridging ligand and Fe ions with a stable 3D framework. The X-ray diffraction (XRD) spectra and X-ray photoelectron spectroscopy (XPS) details demonstrated its structure formed by channels and cavities and a catalysis-promoted electronic environment, which provides a fundamental understanding of this heterogeneous catalyst. Afterwards, it was found that Fe-MMOF can be capable of catalyzing the selective oxidation of versatile natural substrates, acting as an effective peroxidase mimic. The accessibility of the open channels to substrate molecules was also discussed to reveal the steric effect of substrate on catalytic activity. Here, we divided the epoxidation process into three parts, involving formation of HCO4−, the activation of HCO4− to form porMnV = O and the nucleophilic attack of substrate at the electrophilic oxygen of porMnV = O. Remarkably, an insight into the role of HCO3− was gained from the proposed mechanism based on the general idea about homolytic peroxide oxidation reaction.

Dynamic modulation of ANO1/TMEM16A HCO 3 − permeability by Ca 2+ /calmodulin

Anoctamin 1 (ANO1)/transmembrane protein 16A (TMEM16A) is a calcium-activated anion channel that may play a role in HCO(3)(-) secretion in epithelial cells. Here, we report that the anion selectivity of ANO1 is dynamically regulated by the Ca(2+)/calmodulin complex. Whole-cell current measurements in HEK 293T cells indicated that ANO1 becomes highly permeable to HCO(3)(-) at high [Ca(2+)](i). Interestingly, this result was not observed in excised patches, indicating the involvement of cytosolic factors in this process. Further studies revealed that the direct association between ANO1 and calmodulin at high [Ca(2+)](i) is responsible for changes in anion permeability. Calmodulin physically interacted with ANO1 in a [Ca(2+)](i)-dependent manner, and addition of recombinant calmodulin to the cytosolic side of excised patches reversibly increased P(HCO3)/P(Cl). In addition, the high [Ca(2+)](i)-induced increase in HCO(3)(-) permeability was reproduced in mouse submandibular gland acinar cells, in which ANO1 plays a critical role in fluid secretion. These results indicate that the HCO(3)(-) permeability of ANO1 can be dynamically modulated and that ANO1 may play an important role in cellular HCO(3)(-) transport, especially in transepithelial HCO(3)(-) secretion.

Novel Role for Pendrin in Orchestrating Bicarbonate Secretion in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-expressing Airway Serous Cells

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.

Bestrophin-2 mediates bicarbonate transport by goblet cells in mouse colon

Anion transport by the colonic mucosa maintains the hydration and pH of the colonic lumen, and its disruption causes a variety of diarrheal diseases. Cholinergic agonists raise cytosolic Ca2+ levels and stimulate anion secretion, but the mechanisms underlying this effect remain unclear. Cholinergic stimulation of anion secretion may occur via activation of Ca2+-activated Cl- channels (CaCCs) or an increase in the Cl- driving force through CFTR after activation of Ca2+-dependent K+ channels. Here we investigated the role of a candidate CaCC protein, bestrophin-2 (Best2), using Best2-/- mice. Cholinergic stimulation of anion current was greatly reduced in Best2-/- mice, consistent with our proposed role for Best2 as a CaCC. However, immunostaining revealed Best2 localized to the basolateral membrane of mucin-secreting colonic goblet cells, not the apical membrane of Cl--secreting enterocytes. In addition, in the absence of HCO3-, cholinergic-activated current was identical in control and Best2-/- tissue preparations, which suggests that most of the Best2 current was carried by HCO3-. These data delineate an alternative model of cholinergic regulation of colonic anion secretion in which goblet cells play a critical role in HCO3- homeostasis. We therefore propose that Best2 is a HCO3- channel that works in concert with a Cl:HCO3- exchanger in the apical membrane to affect transcellular HCO3- transport. Furthermore, previous models implicating CFTR in cholinergic Cl- secretion may be explained by substantial downregulation of Best2 in Cftr-/- mice.

Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO(3) transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO(3) transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO(3)(-) currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO(3)-dependent pH recovery from a CO(2)-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO(2)/HCO(3)(-)-free solution that was abolished by Cl(-) removal from the bath. In the presence of CO(2)/HCO(3)(-), however, the outward current produced by D555E decreased only slightly after Cl(-) removal. Starting from a Cl(-)-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO(2)/HCO(3)(-). The substitution of Asp(555) with an asparagine also produced a Cl(-) current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO(3)(-). Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp(555) plays a role in HCO(3)(-) selectivity.

Colonic Anion Secretory Defects and Metabolic Acidosis in Mice Lacking the NBC1 [formula omitted] Cotransporter

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.

Effect of Human Carbonic Anhydrase II on the Activity of the Human Electrogenic Na/HCO3 Cotransporter NBCe1-A in Xenopus Oocytes

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I(NBC)) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I(NBC) or the initial rate at which the intracellular pH fell (dpH(i)/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I(NBC) or dpH(i)/dt (Day 4). Because dpH(i)/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I(NBC) slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5' end of NBCe1-A and CA II at the 3' end (EGFP-e1-CAII). We measured I(NBC) or dpH(i)/dt (days 3-4), exposed oocytes to EZA, and measured I(NBC) or dpH(i)/dt (Day 3-4). dpH(i)/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.

HCO3−secretion in the esophageal submucosal glands

The mammalian esophagus has the capacity to secrete a HCO3−and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO3−transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO3−, which averaged 0.21 ± 0.04 μeq·h−1·cm−2. This basal secretion was doubled when stimulated by carbachol but abolished by HCO3−or Cl−removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO3−secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M1cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na+-(HCO3−)n(rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl−/HCO3−exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO3−secretion, a process that involves M1cholinergic receptor stimulation. HCO3−transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO3−secretion is also dependent on serosal Cl−and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.

Renal and intestinal transport defects in Slc26a6-null mice

SLC26A6 (PAT1, CFEX) is an anion exchanger that is expressed on the apical membrane of the kidney proximal tubule and the small intestine. Modes of transport mediated by SLC26A6 include Cl-/formate exchange, Cl-/HCO3- exchange, and Cl-/oxalate exchange. To study its role in kidney and intestinal physiology, gene targeting was used to prepare mice lacking Slc26a6. Homozygous mutant Slc26a6-/- mice appeared healthy and exhibited a normal blood pressure, kidney function, and plasma electrolyte profile. In proximal tubules microperfused with a low-HCO3-/high-Cl- solution, the baseline rate of fluid absorption (Jv), an index of NaCl transport under these conditions, was the same in wild-type and null mice. However, the stimulation of Jv by oxalate observed in wild-type mice was completely abolished in Slc26a6-null mice (P<0.05). Formate stimulation of Jv was partially reduced in null mice, but the difference from the response in wild-type mice did not reach statistical significance. Apical membrane Cl-/base exchange activity, assayed with the pH-sensitive dye BCPCF in microperfused proximal tubules, was decreased by 58% in Slc26a6-/- animals (P<0.001 vs. wild types). In the duodenum, the baseline rate of HCO3- secretion measured in mucosal tissue mounted in Ussing chambers was decreased by approximately 30% (P<0.03), whereas the forskolin-stimulated component of HCO3- secretion was the same in wild-type and Slc26a6-/- mice. We conclude that Slc26a6 mediates oxalate-stimulated NaCl absorption, contributes to apical membrane Cl-/base exchange in the kidney proximal tubule, and also plays an important role in HCO3- secretion in the duodenum.

Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA.

Human NBC3 is an electroneutral Na(+)/HCO(3)(-) cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO(3)(-) metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO(2) + H(2)O left arrow over right arrow HCO(3)(-) + H(+) in many tissues. We investigated whether NBC3, like some Cl(-)/HCO(3)(-) exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K(d) = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 +/- 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 microM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH(i)) recovery rate by 31 +/- 3, or 38 +/- 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH(i) recovery rate by 69 +/- 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [gamma-(32)P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO(3)(-) transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.

Genes Essential to Sodium-dependent Bicarbonate Transport in Cyanobacteria: FUNCTION AND PHYLOGENETIC ANALYSIS

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO(2) uptake systems and two HCO(3)(-) transporters. We transformed a mutant impaired in CO(2) uptake and in cmpA-D encoding a HCO(3)(-)transporter with a transposon inactivation library, and we recovered mutants unable to take up HCO(3)(-) and grow in low CO(2) at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO(2), requires Na(+), and plays the major role in HCO(3)(-) uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na(+)/K(+)-translocating protein) abolished the ability of cells to grow at [Na(+)] higher than 100 mm and severely depressed the activity of the SbtA-mediated HCO(3)(-) transport. We propose that the SbtA-mediated HCO(3)(-) transport is driven by DeltamuNa(+) across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na(+)-dependent HCO(3)(-) transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO(2) supply is limited, or in Prochlorococcus strains that do not possess CO(2) uptake systems or Cmp-dependent HCO(3)(-) transport.

Two CO2 uptake systems in cyanobacteria: four systems for inorganic carbon acquisition in Synechocystis sp. strain PCC6803.

The cyanobacterium Synechocystis sp. strain PCC6803 possesses two CO2 uptake systems; one constitutive, dependent on NdhD3/NdhF3/CupA (Sll1734), and one low-CO2 inducible, dependent on NdhD4/NdhF4/CupB (Slr1302). Homologues of these genes are present in pairs in most cyanobacterial strains. Synechocystis PCC6803 also possesses two types of HCO3- transporters; an ATP-binding cassette (ABC)-type transporter encoded by the cmp operon, and a novel sodium-dependent transporter encoded byslr1512(sbtA) that plays a central role in HCO3- uptake. Mutants impaired for one of these four inorganic-carbon acquisition systems did not show mutant phenotype. Mutants inactivated for both CO2 uptake systems were unable to grow at pH 7.0 in air, although they grew normally at pH 9.0 in air. Additional inactivation of the SbtA-type HCO3- transporter abolished growth at pH 9.0 in air. A fragment containing the promoter region of ndhF3 fused to the coding region of luxAB was inserted into a neutral site of the ΔndhD4 mutant to construct apF3-lux/ ΔndhD4 strain. The luminescence intensity of this strain was low in high-CO2 grown cells, and was increased about 100 times after acclimation to air. Inactivation of the pF3-lux/ ΔndhD4 strain with a transposon-tagging library enabled us to isolate mutants incapable of acclimation to low CO2.