Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pH_i) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl^–/Cl^– and Cl^–/HCO₃^– exchange are inhibited by acid pH_i or extracellular pH (pH_o). 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 pH_i at constant pH_o or varying pH_o. Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal ³⁶Cl^– efflux at pH_o 7.4 and acid shifted the pH_o 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 pH_o sensitivity. In contrast to the effects of DEPC on wild-type AE2, pH_o 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 pH_i, pH_i 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 pH_i and pH_o. pH regulation; histidine residues; Cl^–/HCO₃^– exchange     

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

Human NBC3 is an electroneutral Na⁺/HCO₃^– cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO₃^– metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO₂ + H₂O HCO₃^– + H⁺ in many tissues. We investigated whether NBC3, like some Cl^–/HCO₃^– 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 µM 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 [-³²P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO₃^– transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct. pH regulation; bicarbonate transport; metabolon     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule     

Active Uptake of CO2 by the Diatom Navicula pelliculosa

Mass spectrometry has been used to investigate the transportof CO₂ in the freshwater diatom Navicula pelliculosa. The timecourseof CO₂ formation in the dark after addition of 100 mmol m^–3dissolved inorganic carbon (DIC) to cell suspensions showedthat no external carbonic anhydrase (CA) was present in thesecells. Upon illumination, cells pre-incubated at pH 75 with100 mmol m^–3 DIC, removed almost all free CO₂ from themedium at an initial rate of 285 µmol CO₂ mg^–1Chl h^–1. Equilibrium between HCO₃^– and CO₂ in themedium occurred rapidly upon addition of bovine CA, showingthat CO₂ depletion resulted from a selective uptake of CO₂ ratherthan an uptake of all inorganic carbon species. However, photosyntheticO₂ evolution rate remained constant after CO₂ had been depletedfrom the medium indicating that photosynthesis is sustainedprimarily by active HCO₃^– uptake. Treatment of cells with2-iodoacetamide (83 mol m^–3) completely inhibited CO₂fixation but had little effect on CO₂ transport since initialrates of CO₂ depletion were about 81% that of untreated cells.Transfer of iodoacetamide-treated cells to the dark caused arapid increase in the CO₂ concentration in the medium largelydue to the efflux of the unfixed intracellular DIC pool whichwas found to be about 194 times the concentration of that inthe external medium. These results indicate that Navicula pelliculosaactively takes up molecular CO₂ against a concentration gradientby a process distinct from HCO₃^– transport. Key words: Dissolved inorganic carbon, carbonic anhydrase, bicarbonate transport, CO₂ transport, mass spectrometry     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

The abts and sulp families of anion transporters from Caenorhabditis elegans

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.     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat     

The functional and physical relationship between the DRA bicarbonate transporter and carbonic anhydrase II

COOH-terminal cytoplasmic tails ofchloride/bicarbonate anion exchangers (AE) bind cytosolic carbonicanhydrase II (CAII) to form a bicarbonate transport metabolon, amembrane protein complex that accelerates transmembrane bicarbonateflux. To determine whether interaction with CAII affects thedownregulated in adenoma (DRA) chloride/bicarbonate exchanger, anionexchange activity of DRA-transfected HEK-293 cells was monitored byfollowing changes in intracellular pH associated with bicarbonatetransport. DRA-mediated bicarbonate transport activity of 18 ± 1 mM H⁺ equivalents/min was inhibited 53 ± 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by themembrane-impermeant carbonic anhydrase inhibitor,1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactiveCAII mutant, V143Y, reduced AE1 transport activity by 61 ± 4%without effect on DRA transport activity (105 ± 7% transport activity relative to DRA alone). We conclude that cytosolic CAII isrequired for full DRA-mediated bicarbonate transport. However, DRAdiffers from other bicarbonate transport proteins because its transportactivity is not stimulated by direct interaction with CAII.

     

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl−/HCO3− exchanger,AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO₂ to HCO₃^?+H⁺. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl^?/HCO₃^? exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl^?/NO₃^? exchange assays, which were independent of CA activity, and in Cl^?/HCO₃^? exchange assays. Transport was measured by following changes of intracellular [Cl^?] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl^?/NO₃^? exchange activity with EC₅₀ values in the range 0.22–2.8 μM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 μM of each compound was only 22–53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl^?/HCO₃^? exchange assays, which depend on functional CA to produce transport substrate, 40 μM celecoxib inhibited AE1 by 62±4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.     

The Location of the Transporting System for Inorganic Carbon and the Nature of the Form Translocated in Chlamydomonas reinhardtii

The permeability of the plasmalemma of Chlamydomonas reinhardtiicells was increased by treatment with poly-L-lysine or dimethylsulphoxideas indicated by 3-phosphoglyceric acid dependent O₂ evolution.These treatments decreased the ability of the cells to accumulateinorganic carbon internally and hence their photosynthetic affinityfor inorganic carbon in the medium. With saturating light andinorganic carbon, the photosynthetic rate was less affectedby the poly-L-lysine and dimethylsulphoxide treatments. Thusthe poly-L-lysine and dimethylsulphoxide did not alter the activityof the chloroplasts but rather made the intracellular inorganiccarbon pool more freely exchangeable with the medium. It isconcluded that the transporting system for inorganic carbonis located at the plasmalemma. Treatment with Diamox, an inhibitor of carbonic anhydrase, didnot affect photosynthetic rate and accumulation of inorganiccarbon when CO₂ was supplied but strongly inhibited both parameterswhen HCO^–₃ was supplied. In a mutant of Chlamydomonasreinhardtii lacking a cell wall, carbonic anhydrase leaks tothe medium and uptake of inorganic carbon is much faster whenCO₂ is supplied than when HCO^–₃ is supplied. These resultssuggest that CO₂ rather than HCO^–₃ is the inorganic carbonspecies that is actively translocated across the plasmalemma. Key words: Chlamydomonas, Inorganic carbon uptake     

The alpha2-adrenergic receptor agonist UK 14,304 inhibits secretin-stimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats

Secretin stimulates ductal secretion by activation of cAMP PKA CFTR Cl^–/HCO₃^– exchanger in cholangiocytes. We evaluated the expression of _2A-, _2B-, and _2C-adrenergic receptors in cholangiocytes and the effects of the selective ₂-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na⁺/H⁺ exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl^– efflux, and Cl^–/HCO₃^– exchanger activity in purified cholangiocytes. ₂-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl^– efflux, and Cl^–/HCO₃^– exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. ₂-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases. bicarbonate secretion; chloride efflux; gastrointestinal hormones; intrahepatic biliary epithelium; protein kinase A     

Transport and Fixation of Inorganic Carbon in the Cells of Chlorella vulgaris 11h Grown under Ordinary Air

Intracellular accumulation of inorganic carbon (C_i) and itsfixation in photosynthesis were investigated using siliconeoil layer filtering centrifugation technique with the cellsof Chlorella vulgaris 11h grown under ordinary air. Both CO₂and HCO₃^– were transported into the cells from the reactionmedium and accumulated in the cells, but the rate of transportwas much faster for the former than the latter. ¹⁴C-fixationfrom the total transported C_i was much more efficient when CO₂was added in the external medium than when HCO₃^– was added.This indicates that CO₂ and HCO₃^– were not converted tothe common compound in the cells during the initial period ofphotosynthesis. Accumulation of Cⁱ into the cells was much lesssusceptible to low temperature than its fixation. Accumulationof Cⁱ was also observed in the dark. Ethoxyzolamide, an inhibitorof carbonic anhydrase (CA), inhibited the fixation of accumulatedCO₂ in the cells, suggesting that CA enhanced the supply ofCO₂ to the reaction site of ribulose bisphosphate carboxylasein the stroma. Mechanism for transport and fixation of Cⁱ duringphotosynthesis in low-CO₂ cells of C. vulgaris 1lh was proposedfrom these results. (Received March 19, 1986; Accepted June 26, 1986)     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

Transport and Fixation of Inorganic Carbon during Photosynthesis in Cells of Anabaena Grown under Ordinary Air: III. Some Characteristics of the HCO3--Transport System in Cells Grown under Ordinary Air

In cells of cyanobacterium Anabaena variabilis grown under ordinaryair (low-CO₂ cells), the transport of both CO₂ and HCO₃^–was significantly enhanced by Na⁺. This effect was pronouncedas the external pH increased. When low-CO₂ cells were treatedwith an inhibitor of carbonic anhydrase (CA), only CO₂ transportbut not HCO₃^– transport, was inhibited. The initial rateof photosynthetic carbon fixation as a function of the concentrationof internal inorganic carbon (IC) was practically the same irrespectiveof whether CO₂ or HCO₃^– was externally supplied. Theseresults suggest that IC is actively transported through theplasma membrane in a form of HCO₃^– probably by some transporterand that the transmembrane Na⁺ gradient is involved in thisIC transport system. Free CO₂ may be hydrated by CA to HCO₃^–and then transported to the cells by this transporter. On the other hand, CO₂ is actively taken up by cells grown withair containing 5% CO₂ (high-CO₂ cells) though the enhancingeffect of Na⁺ was much smaller in high- CO₂ cells than in low-CO₂cells. The initial rate of fixation as a function of internal IC concentrationindicated that the rate of the carboxylation reaction of accumulatedIC is higher in I0W-CO₂ cells than in high-CO₂ cells. The studieswith ethoxyzolamide indicated that even in low-CO₂ cells, CAdoes not function inside Anabaena cells. These results suggestthat inside the low-CO₂ cells of Anabaena, some mediator(s)facilitates the transport of IC to RuBPCase. (Received January 23, 1987; Accepted April 24, 1987)     

The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl-/HCO3- exchanger binds carbonic anhydrase IV

Cytosolic carbonic anhydrase II (CAII) and the cytoplasmic C-terminal tails of chloride/bicarbonate anion exchange (AE) proteins associate to form a bicarbonate transport metabolon, which maximizes the bicarbonate transport rate. To determine whether cell surface-anchored carbonic anhydrase IV (CAIV) interacts with AE proteins to accelerate the bicarbonate transport rate, AE1-mediated bicarbonate transport was monitored in transfected HEK293 cells. Expression of the inactive CAII V143Y mutant blocked the interaction between endogenous cytosolic CAII and AE1, AE2, and AE3 and inhibited their transport activity (53 +/- 3, 49 +/- 10, and 35 +/- 1% inhibition, respectively). However, in the presence of V143Y CAII, expression of CAIV restored full functional activity to AE1, AE2, and AE3 (AE1, 101 +/- 3; AE2, 85 +/- 5; AE3, 108 +/- 1%). In Triton X-100 extracts of transfected HEK293 cells, resolved by sucrose gradient ultracentrifugation, CAIV recruitment to the position of AE1 suggested a physical interaction between CAIV and AE1. Gel overlay assays showed a specific interaction between CAIV and AE1, AE2, and AE3. Glutathione S-transferase pull-down assays revealed that the interaction between CAIV and AE1 occurs on the large fourth extracellular loop of AE1. We conclude that AE1 and CAIV interact on extracellular loop 4 of AE1, forming the extracellular component of a bicarbonate transport metabolon, which accelerates the rate of AE-mediated bicarbonate transport.     

Photosynthetic Electron Transport under Phosphorylating Conditions as Influenced by Different Concentrations of Various Salts

Osman, M. E-A. H. and El-Shentenawy, F. 1988. Photosyntheticelectron transport under phosphorylating conditions as influencedby different concentrations of various salts.—J. exp.Bot. 39: 859–863. The rate of light-induced electron transport by isolated spinachthylakoids under phosphorylating conditions, as affected bydifferent concentrations of Br^–, Cl^–, NO₃^–,HCO₃^–, SO₄^2– and CO₃^2– has been investigated.The results show that both low and high concentrations of HCO₃^2–stimulated the oxygen evolution capacity under phosphorylatingconditions, whereas only low concentrations of CO₃^2–,SO₄^2– and Cl^– stimulated the oxygen evolution capacity.However, irrespective of concentration, both Br^– and NO₃^–reduced this capacity. The rate of photosynthetic electron transportwas generally stimulated by addition of ADP, even in cases whereelectron transport was inhibited by addition of bromide andnitrate. The different concentrations of these anions also causedreduction of the power generated by proton pumping and usedfor phosphorylation. The greatest level of reduction was observedin the presence of high concentrations of Cl^– and HCO₃^–. Key words: Spinach thylakoids, photosynthetic electron transport, phosphorylation     

Kinetic Studies on the Active Species of Inorganic Carbon Absorbed by the Cells of Dunaliella tertiolecta

Cells of Dunaliella tertiolecta which had been grown in ordinaryair (low-CO₂ cells) had high carbonic anhydrase (CA) activityon the cell surface and mainly utilized HCO₃^– for photosynthesis.When CA activity on the cell surface was inhibited by Diamoxor subtilisin, the cells utilized CO₂. When bovine CA was added,the subtilisin-treated low-CO₂ cells utilized mainly HCO₃^–.When grown in air containing 2% CO₂, the cells had low CA activityon the cell surface, and preferred CO₂ to HCO₃^–. Kineticanalysis of these results indicated that low-CO₂ cells of D.tertiolecta absorb CO₂ which was converted from HCO₃^–via the CA located on the cell surface. (Received June 29, 1985; Accepted October 9, 1985)     

Further Characteristics of Salt-Dependent Bicarbonate Use by the Seagrass Zostera muelleri

Millhouse, J. and Strother, S. 1987. Further characteristicsof salt-dependent bicarbonate use by the seagrass Zostera muelleri.—J.exp. Bot. 38: 1055–1068. The contribution of HCO^–₃to photosynthetic O₂ evolutionin the seagrass Zostera muelleri Irmisch ex Aschers. increasedwith increasing salinity of the bathing seawater when the inorganiccarbon concentration was kept constant. K_1/2 (seawater salts)for HCO^–₃ -dependent photosynthesis was 66% of seawatersalinity. Both short- and long-term pretreatment at low salinitiesstimulated photosynthesis in full strength seawater. Twentyfour hours pre-incubation of seagrass plants in 3·0 molm^–3 NaHCO₃ resulted in increased photosynthesis at allsalinities, apparently due to stimulation of HCO^–₃ use(K_1/2 (seawater salts) = 26%). V_max (HCO^–₃) was not affectedby low salinity pretreatment. The kinetics of HCO^–₃ stimulationby the major seawater cations was investigated. Ca²⁺ was themost effective cation with the highest V_max (HCO^–₃) andwith K_1/2(Ca²⁺) = 14 mol m^–3. Mg²⁺ was also very effectiveat less than 50 mol m^–3 but higher concentrations wereinhibitory. This inhibition cannot be accounted for solely byprecipitation of MgCO₃. Na⁺ and K⁺ were both capable of stimulatingHCO^–₃ use. Stimulation was in two distinct parts. Up to500 mol m^–3, both citrate and chloride salts gave similarresults (K_1/2(Na⁺) 81 mol m^–3, V_max(HCO^–₃) 0·26µmol O₂ mg^–1 chl min^–1), but use of citratesalts above 500 mol m^–2 caused a second stimulation ofHCO^–₃ use (K_1/2(Na⁺) 830 mol m^–3, V_max(HCO^–₃)0·68 µmol O₂ mg^–1 chl min^–1). V_max(HCO^–₃)for the second-phase Na⁺ or K⁺ stimulation was of the same orderas for Ca²⁺-stimulated HCO^–₃ use. To further characterizesalt-dependent HCO^–₃ use, the sensitivity of photosynthesisto Tris and TES buffers was investigated. The effects of Trisappear to be due to the action of Tris⁺ causing stimulationof HCO^–₃ -dependent photosynthesis in the absence of salt,but inhibition of HCO^–₃ use in saline media. TES has noeffect on photosynthesis. External carbonic anhydrase, althoughimplicated in salt-dependent HCO^–₃ use in Z. muelleri,could not be detected in whole leaves. Key words: Zostera muelleri, HCO^–₃ use, salinity     

The Role of Carbonic Anhydrase in Blood Ion and Acid-Base Regulation

The role of carbonic anhydrase (CA) in ion transport processesof aquatic and terrestrial arthropod species is reviewed. Inboth insects and crustaceans CA is found in a variety of iontransporting tissues. The bulk of CA activity in crustaceansis concentrated in the posterior gills, which are morphologicallyand biochemically adapted for ion transport. The enzyme canbe specifically localized to gill lamellae which contain largepopulations of salt transporting chloride cells. Enzyme activityin the posterior gills of species having the ability to regulateblood ion concentrations increases when these organisms areacclimated to environmental salinities in which they ion regulate.In stenohaline, ion conforming species branchial CA activityis uniformly low, being only 5–10% that in regulatingspecies. Studies on the blue crab, Callinectes sapidus, usingthe specific CA inhibitor acetazolamide have shown that theenzyme is indeed important in blood ion regulation. Blood Na^$and Cl^– concentrations are both severely lowered in drug-treatedanimals acclimated to low salinity, while they remain virtuallyunaffected in animals acclimated to high salinity, in whichthe animal is an ion conformer. High salinity acclimated crabstreated with acetazolamide do not survive transfer to low salinity,and mortality is related to a breakdown in the ion regulatorymechanism. Branchial CA most likely functions in the hydrationof respiratory CO₂ to H^$ and HCO₃^–, which serve as counterionsfor the active uptake of Na^$ and Cl^–, respectively. Interrestrial species the role of CA is unclear and merits furtherinvestigation.     

Effects of Ammonia and Methylamine on Cl- Transport and on the pH Changes and Circulating Electric Currents Associated with HCO3- Assimilation in Chara corallina

Low concentrations of ammonia and methylamine greatly increaseCl^– influx into Chara corallina. Both amines have theirmaximum effect at pH 6.5–7.5. The amine stimulation ofCl^– influx is small below about pH 5.5. Above pH 8.5 theremay be inhibition of influx by amines. Concentrations of 10–25µM ammonia are sufficient to cause the maximum stimulationof Cl^– influx; the corresponding methylamine concentrationsare 0.1–0.2 mM. It is concluded that entry of amine cations(NH₄^$ and CH₃NH₃^$), rather than unionized bases (NH₃ and CH₃NH₂),causes Cl^– transport to be increased. Increases in rates of Cl^– transport are not necessarilyaccompanied by effects on HCO₃^$ assimilation and OH^– efflux.Measurements of localized pH differences at the cell surfaceand of circulating electric currents in the bathing solutionshow that these phenomena are only significantly affected byammonia at or above 50 µM and by methylamine at or above1.0 mM. The significance of the effects of amines is assessedin relation to current ideas about transport of Cl^–, HCO₃^–,and OH^–.