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

2002 ◽  
Vol 283 (5) ◽  
pp. C1522-C1529 ◽  
Author(s):  
Deborah Sterling ◽  
Nathan J. D. Brown ◽  
Claudiu T. Supuran ◽  
Joseph R. Casey
Keyword(s):  
Carbonic Anhydrase ◽  
Direct Interaction ◽  
Carbonic Anhydrase Ii ◽  
Bicarbonate Transport ◽  
Transport Activity ◽  
Physical Relationship ◽  
Hek 293 ◽  
Bicarbonate Transporter ◽  
293 Cells ◽  
Cytoplasmic Tails

COOH-terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA-transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. 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 the membrane-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 inactive CAII 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 is required for full DRA-mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII.

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

2004 ◽  
Vol 286 (6) ◽  
pp. C1423-C1433 ◽  
Author(s):  
Frederick B. Loiselle ◽  
Patricio E. Morgan ◽  
Bernardo V. Alvarez ◽  
Joseph R. Casey
Keyword(s):  
Carbonic Anhydrase ◽  
Recovery Rate ◽  
Dominant Negative ◽  
Carbonic Anhydrase Ii ◽  
Transport Activity ◽  
Wild Type ◽  
Hek 293 ◽  
Transfected Cells ◽  
293 Cells ◽  
Optimal Function

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 Kd = 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 (pHi) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pHi 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 [γ-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.

Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters

2007 ◽  
Vol 293 (2) ◽  
pp. C738-C748 ◽  
Author(s):  
Patricio E. Morgan ◽  
Silvia Pastoreková ◽  
Alan K. Stuart-Tilley ◽  
Seth L. Alper ◽  
Joseph R. Casey
Keyword(s):  
Gastric Mucosa ◽  
Carbonic Anhydrase ◽  
Catalytic Domain ◽  
Hek293 Cells ◽  
Bicarbonate Transport ◽  
Physical Interaction ◽  
Human Gastric Mucosa ◽  
Bicarbonate Transporter ◽  
Bicarbonate Transporters ◽  
Physical Interactions

Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO2-HCO3−conversions and coupled HCO3−transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl−/HCO3−exchanger AE2 or the putative Cl−/HCO3−exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl−/HCO3−exchangers to form a bicarbonate transport metabolon.

scholarly journals Human geneSLC41A1encodes for the Na+/Mg2+exchanger

2012 ◽  
Vol 302 (1) ◽  
pp. C318-C326 ◽  
Author(s):  
Martin Kolisek ◽  
Axel Nestler ◽  
Jürgen Vormann ◽  
Monika Schweigel-Röntgen
Keyword(s):  
Mammalian Cells ◽  
Molecular Level ◽  
Transport Activity ◽  
Human Embryonic Kidney ◽  
Efflux System ◽  
Dependent Protein Kinase ◽  
Complete Replacement ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Protein

Magnesium (Mg2+), the second most abundant divalent intracellular cation, is involved in the vast majority of intracellular processes, including the synthesis of nucleic acids, proteins, and energy metabolism. The concentration of intracellular free Mg2+([Mg2+]i) in mammalian cells is therefore tightly regulated to its optimum, mainly by an exchange of intracellular Mg2+for extracellular Na+. Despite the importance of this process for cellular Mg2+homeostasis, the gene(s) encoding for the functional Na+/Mg2+exchanger is (are) still unknown. Here, using the fluorescent probe mag-fura 2 to measure [Mg2+]ichanges, we examine Mg2+extrusion from hSLC41A1-overexpressing human embryonic kidney (HEK)-293 cells. A three- to fourfold elevation of [Mg2+]iwas accompanied by a five- to ninefold increase of Mg2+efflux. The latter was strictly dependent on extracellular Na+and reduced by 91% after complete replacement of Na+with N-methyl-d-glucamine. Imipramine and quinidine, known unspecific Na+/Mg2+exchanger inhibitors, led to a strong 88% to 100% inhibition of hSLC41A1-related Mg2+extrusion. In addition, our data show regulation of the transport activity via phosphorylation by cAMP-dependent protein kinase A. As these are the typical characteristics of a Na+/Mg2+exchanger, we conclude that the human SLC41A1 gene encodes for the Na+/Mg2+exchanger, the predominant Mg2+efflux system. Based on this finding, the analysis of Na+/Mg2+exchanger regulation and its involvement in the pathogenesis of diseases such as Parkinson's disease and hypertension at the molecular level should now be possible.

Characterization of an epilepsy-associated variant of the human Cl−/HCO3−exchanger AE3

2009 ◽  
Vol 297 (3) ◽  
pp. C526-C536 ◽  
Author(s):  
Gonzalo L. Vilas ◽  
Danielle E. Johnson ◽  
Paul Freund ◽  
Joseph R. Casey
Keyword(s):  
Protein Trafficking ◽  
Specific Inhibitor ◽  
Similar Degree ◽  
Transport Activity ◽  
Wild Type ◽  
Hek 293 ◽  
293 Cells ◽  
The Brain ◽  
Exchange Activity

Anion exchanger 3 (AE3), expressed in the brain, heart, and retina, extrudes intracellular HCO3−in exchange for extracellular Cl−. The SLC4A3 gene encodes two variants of AE3, brain or full-length AE3 (AE3fl) and cardiac AE3 (cAE3). Epilepsy is a heterogeneous group of disorders characterized by recurrent unprovoked seizures that affect about 50 million people worldwide. The AE3-A867D allele in humans has been associated with the development of IGE (IGE), which accounts for ∼30% of all epilepsies. To examine the molecular basis for the association of the A867D allele with IGE, we characterized wild-type (WT) and AE3fl-A867D in transfected human embryonic kidney (HEK)-293 cells. AE3fl-A867D had significantly reduced transport activity relative to WT (54 ± 4%, P < 0.01). Differences in expression levels or the degree of protein trafficking to the plasma membrane did not account for the defect of AE3fl-A867D. Treatment with 8-bromo-cAMP (8-Br-cAMP) increased Cl−/HCO3−exchange activity of WT and AE3fl-A867D to a similar degree, which was abolished by preincubation with the protein kinase A (PKA)-specific inhibitor H89. This indicates that PKA regulates WT and AE3fl-A867D Cl−/HCO3−exchange activity. No difference in Cl−/HCO3−exchange activity was found between cultures of mixed populations of neonatal hippocampal cells from WT and slc4a3−/−mice. We conclude that the A867D allele is a functional (catalytic) mutant of AE3 and that the decreased activity of AE3fl-A867D may cause changes in cell volume and abnormal intracellular pH. In the brain, these alterations may promote neuron hyperexcitability and the generation of seizures.

scholarly journals Transport activity of AE3 chloride/bicarbonate anion-exchange proteins and their regulation by intracellular pH

1999 ◽  
Vol 344 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Deborah STERLING ◽  
Joseph R. CASEY
Keyword(s):  
Cell Surface ◽  
Anion Exchange ◽  
Intracellular Ph ◽  
Chloride Concentration ◽  
Full Length ◽  
Transport Activity ◽  
Hek 293 ◽  
293 Cells ◽  
Wide Range ◽  
Acid Loading

Plasma membrane Cl-/HCO3- anion-exchange (AE) proteins contribute to regulation of intracellular pH (pHi). We characterized the transport activity and regulation by pHi of full-length AE3 and the cardiac isoform, AE3c, both of which are expressed in the heart. AE3c is an N-terminal variant of AE3. We also characterized AE1, AE2 and a deletion construct (AE3tr) coding for the common region of AE3 and AE3c. AE proteins were expressed by transient transfection of HEK-293 cells, and transport activity was monitored by following changes of intracellular pH or intracellular chloride concentration associated with anion exchange. Transport activities, measured as proton flux (mM H+˙min-1), were as follows: AE1, 24; AE2, 32; full-length AE3, 9; AE3c, 4 and AE3tr, 4. The wide range of transport activities is not explained by variation of cell surface processing since approx. 30% of each isoform was expressed on the cell surface. pHi was clamped at a range of values from 6.0-9.0 to examine regulation of AE proteins by pHi. Whereas AE2 was steeply inhibited by acid pHi, AE1, AE3 and AE3c were essentially insensitive to changes of pHi. We conclude that AE3 and AE3c can contribute to pHi recovery after cellular-acid loading.

scholarly journals A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sina Ibne Noor ◽  
Somayeh Jamali ◽  
Samantha Ames ◽  
Silke Langer ◽  
Joachim W Deitmer ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Xenopus Oocytes ◽  
Proton Exchange ◽  
Monocarboxylate Transporter ◽  
Carbonic Anhydrase Ii ◽  
Transport Activity ◽  
Proton Shuttling ◽  
Mcf 7

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme’s intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.

scholarly journals Molecular mechanism underlying transport and allosteric inhibition of bicarbonate transporter SbtA

2021 ◽  
Vol 118 (22) ◽  
pp. e2101632118
Author(s):  
Sunzhenhe Fang ◽  
Xiaowei Huang ◽  
Xue Zhang ◽  
Minhua Zhang ◽  
Yahui Hao ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Three Dimensional ◽  
Underlying Mechanism ◽  
Bicarbonate Transport ◽  
Transport Activity ◽  
Allosteric Inhibition ◽  
Core Domain ◽  
Bicarbonate Transporter ◽  
Sodium Dependent ◽  
Loop Conformation

SbtA is a high-affinity, sodium-dependent bicarbonate transporter found in the cyanobacterial CO2-concentrating mechanism (CCM). SbtA forms a complex with SbtB, while SbtB allosterically regulates the transport activity of SbtA by binding with adenyl nucleotides. The underlying mechanism of transport and regulation of SbtA is largely unknown. In this study, we report the three-dimensional structures of the cyanobacterial Synechocystis sp. PCC 6803 SbtA–SbtB complex in both the presence and absence of HCO3− and/or AMP at 2.7 Å and 3.2 Å resolution. An analysis of the inward-facing state of the SbtA structure reveals the HCO3−/Na+ binding site, providing evidence for the functional unit as a trimer. A structural comparison found that SbtA adopts an elevator mechanism for bicarbonate transport. A structure-based analysis revealed that the allosteric inhibition of SbtA by SbtB occurs mainly through the T-loop of SbtB, which binds to both the core domain and the scaffold domain of SbtA and locks it in an inward-facing state. T-loop conformation is stabilized by the AMP molecules binding at the SbtB trimer interfaces and may be adjusted by other adenyl nucleotides. The unique regulatory mechanism of SbtA by SbtB makes it important to study inorganic carbon uptake systems in CCM, which can be used to modify photosynthesis in crops.

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter

2008 ◽  
Vol 294 (4) ◽  
pp. C1074-C1078 ◽  
Author(s):  
Takuya Matsumoto ◽  
Takuji Kanamoto ◽  
Masato Otsuka ◽  
Hiroshi Omote ◽  
Yoshinori Moriyama
Keyword(s):  
Organic Cation ◽  
Ph Dependence ◽  
Substrate Recognition ◽  
Transport Activity ◽  
Hek 293 ◽  
Mutant Proteins ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Transmembrane Regions

Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H+/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in Vmax value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.

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