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 Transport activity of chimaeric AE2-AE3 chloride/bicarbonate anion exchange proteins

2003 ◽  
Vol 371 (3) ◽  
pp. 687-696 ◽  
Author(s):  
Jocelyne FUJINAGA ◽  
Frederick B. LOISELLE ◽  
Joseph R. CASEY
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cytoplasmic Domain ◽  
Anion Transport ◽  
Transport Activity ◽  
Surface Processing ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Anion Transport Activity

Chloride/bicarbonate anion exchangers (AEs), found in the plasma membrane of most mammalian cells, are involved in pH regulation and bicarbonate metabolism. Although AE2 and AE3 are highly similar in sequence, AE2-transport activity was 10-fold higher than AE3 (41 versus 4 mM · min−1 respectively), when expressed by transient transfection of HEK-293 cells. AE2–AE3 chimaeras were constructed to define the region responsible for differences in transport activity. The level of AE2 expression was approx. 30% higher than that of AE3. Processing to the cell surface, studied by chemical labelling and confocal microscopy, showed that AE2 is processed to the cell surface approx. 8-fold more efficiently than AE3. The efficiency of cell-surface processing was dependent on the cytoplasmic domain, since the AE2 domain conferred efficient processing upon the AE3 membrane domain, with a predominant role for amino acids 322–677 of AE2. AE2 that was expressed in HEK-293 cells was glycosylated, but little of AE3 was. However, AE2 expressed in the presence of the glycosylation inhibitor, tunicamycin, was not glycosylated, yet retained 85 ± 8% of anion-transport activity. Therefore glycosylation has little, if any, role in the cell-surface processing or activity of AE2 or AE3. We conclude that the low anion-transport activity of AE3 in HEK-293 cells is due to low level processing to the plasma membrane, possibly owing to protein interactions with the AE3 cytoplasmic domain.

scholarly journals Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface

2004 ◽  
Vol 378 (3) ◽  
pp. 1015-1021 ◽  
Author(s):  
Joanne C. CHEUNG ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Palmitic Acid ◽  
Anion Exchanger ◽  
Cell Surface Expression ◽  
Co2 Removal ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

Nucleotidase Activity of CD39/NTPDase1 Is Dependent on Internal Proteolytic Cleavage Which Contributes to Lipid Raft Localization.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3634-3634
Author(s):  
Kim E. Olson ◽  
Marinus Johan Broekman ◽  
Ashley E. Olson ◽  
Dianne Pulte ◽  
Aaron J. Marcus
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Enzymatic Activity ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Purinergic Signaling ◽  
Full Length ◽  
293 Cells ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Abstract Brief trypsin exposure increases apyrase activity in hCD39 expressing cells, as previously reported (Schulte am Esch et al, Biochemistry38:2248, 1999). Since regulated proteolytic cleavage of CD39 would allow for a rapid response to extracellular stimuli, we studied the relationship between observed CD39 cleavage and enzymatic activity. We generated N- and C-terminal VP16-tagged hCD39 to study CD39 expression, processing, and activity in transiently transfected HEK 293 cells. We found that optimal enzymatic activity of CD39 indeed depends on incorporation into cholesterol-rich plasma membrane domains (lipid "rafts"). Membrane fractions from hCD39 -transfected 293 cells readily hydrolyze ATP. Pretreatment of 293 cells with the cholesterol-depleting agent methyl β cyclodextrin (MBCD) results in a dose-dependent decrease in ATPase activity. In addition, treatment of isolated membranes with MBCD also decreases enzymatic activity. We next performed Western blot analyses of membranes prepared from hCD39-transfected 293 cells treated with membrane-impermeant crosslinking agents. These experiments demonstrated a dose-dependent, MBCD-reversible decrease in monomeric CD39. Taken together, these data demonstrate that CD39 enzyme activity resides in raft-localized CD39. Western blots of membrane fractions from cells transfected with N- or C-terminal VP16-tagged hCD39 show partial cleavage of full-length CD39 to yield a 20kDa N-terminal and 50 kDa C-terminal fragments. Biotinylation studies established that both fragments are expressed on the cell surface. As with full-length CD39, crosslinking results in dose-dependent decreases of both monomeric species. Moreover, prior cholesterol depletion with MBCD abolishes crosslinking. Since the cleavage products of full-length CD39 are expressed on the cell surface and localize to lipid rafts, we examined the relation between CD39 cleavage, ATPase activity and lipid raft localization using a panel of cell permeable protease inhibitors. 293 cells transfected with N-terminal VP16-tagged CD39 were treated with AEBSF (serine protease inhibitor), zYVAD.fmk (caspase inhibitor), zLLY.fmk (calpain inhibitor) or the furin inhibitor Furin I. All inhibitors resulted in dose-dependent decreases in formation of the VP16-tagged N-terminal fragment. Concomitantly, ATPase assays of the membrane fractions demonstrated a corresponding dose-dependent decrease in enzymatic activity. Finally, we established that CD39 cleavage promotes raft localization, since protease inhibition decreased the fraction of CD39 susceptible to crosslinking with all inhibitors tested. In summary, we have established that generation of optimally active, raft-localized CD39 requires prior limited proteolysis of the full-length molecule. Activation of caspase-1 by exposure of cells to ATP leads to processing and release of interleukin family members. We propose that purinergic signaling might also enhance CD39 cleavage in vascular cells by an as yet unidentified protease. Our data suggest that subsequent increased cell surface apyrase activity leads to dampening of purinergic signaling and a resulting increase in antithrombotic activity. Of note, we identified an alternately spliced isoform of CD39 which inhibits cleavage of the full-length molecule.

Assembly of Active High Molecular Weight CD39 Complex: Role of Proteolysis and Endocytosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2110-2110
Author(s):  
Kim Olson ◽  
Aaron j Marcus
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Enzymatic Activity ◽  
Sucrose Gradient ◽  
Early Endosome ◽  
Full Length ◽  
Membrane Cholesterol ◽  
293 Cells ◽  
Proportional Decrease

Abstract Abstract 2110 HUVECs, neutrophils, monocyte/macrophages and T cell subsets all express ectonucleoside triphosphate diphosphohydrolase 1 (CD39) on the cell surface. In addition, they all express both P2X and P2Y receptors and dose dependently respond to ATP. ATP enhances superoxide production in activated neutrophils and supports chemotaxis of macrophages responding to a chemoattractant. ATP also induces apoptosis of anti-inflammatory T regulatory cells and supports the differentiation of pro-inflammatory Th17 cells. Lastly, ADP drives thrombus formation by activation of platelet P2Y12 receptors. An increase in the activity of expressed CD39 would result in increased metabolism of these pro-thrombotic and pro-inflammatory nucleotides. We examined the reported relationship between CD39 cleavage and cell surface enzymatic activity. We cloned N-terminal and C-terminal V5 and VP16 tagged CD39 into eukaryotic expression vectors as well as lentiviral genomic vectors for analysis of CD39 expression in 293 cells and HUVECs. Western blots of membrane fractions prepared from HUVECs and transiently and stably transfected 293 cells identified the previously described CD39 cleavage fragments. Streptavidin precipitation of biotinylated membrane proteins demonstrated that both fragments are present on the cell surface of both transduced HUVECs and transfected 293 cells. We next separated membranes on a discontinuous sucrose gradient to yield ER, Golgi/plasma membrane, early endosome and late endosome enriched membrane fractions. Surprisingly, full length CD39 and the N- and C-terminal fragments appeared in both the Golgi/plasma membrane and early endosome fractions. In addition, the early endosome CD39 exhibited enzyme activity equal to that of the Golgi/plasma membrane CD39. We then prepared membranes from cells treated with either chloroquine or bafilomycin, reagents known to interfere with endosomal acidification and/or maturation. In each case, we observed a decrease in the fractional cleavage of full length CD39 and a proportional decrease in associated enzymatic activity. When sonicated membranes were resolved on a continuous sucrose gradient, the N- and C-terminal fragments and a fraction of full length CD39 as well as maximal enzymatic activity were found in the low density, “raft” fractions. These results suggest that the formation of an enzymatically active CD39 complex requires N- and C-terminal CD39 fragments as well as membrane cholesterol. When 293 cells that stably expressed CD39 were transiently transfected with dominant negative dynamin 2, we observed a decrease in fractional cleavage as well as a proportional decrease in enzymatic activity. This result suggested that the cleavage event occurs following endocytosis of plasma membrane expressed CD39. Finally, we treated both stably transfected 293 cells and HUVECs with the cell permeable cysteine protease inhibitor zLLY.fmk. Prepared membranes analyzed by Western blot showed a decrease in fractional cleavage of full length CD39. Apyrase assays showed a corresponding decrease in ATPase and ADPase activity. We then examined the cholesterol dependence of CD39 activity by depleting membrane cholesterol with MβCD. As expected, ATPase activity decreased in a dose dependent manner. The predominant “active” species appeared as a 1.4 megadalton complex on a 3–12% BN gel of Digitonin solubilized membranes prepared from cultured cells treated with the cleavable cross-linker DTSSP. As a result of membrane cholesterol depletion, there was a proportional decrease in the amount of full length CD39 and N- and C-terminal fragments present in the DTSSP cross-linked HMW complex. Interestingly, there was a marked increase in the abundance of lower molecular weight complexes in cholesterol depleted cells. In conclusion, we provide evidence that CD39 enzymatic activity resides in a megadalton complex formed by protein-protein interactions between full length CD39 and C- and N-terminal fragments generated by cleavage of the full length molecule. Assembly of the oligomeric protein complex requires membrane cholesterol and likely occurs on intracellular membranes. Moreover, approximately 50% of the active enzyme complex remains sequestered on intracellular membranes. These results suggest that up-regulation of CD39 metabolism of pro-thrombotic and pro-inflammatory nucleotides involves pathways independent of gene transcription. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

2002 ◽  
Vol 157 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
László Hunyady ◽  
Albert J. Baukal ◽  
Zsuzsanna Gáborik ◽  
Jesus A. Olivares-Reyes ◽  
Márta Bor ◽  
...  
Keyword(s):  
Cell Surface ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Slow Component ◽  
Ang Ii ◽  
Hek 293 ◽  
Recycling Endosomes ◽  
293 Cells ◽  
Early Endosomes ◽  
Green Fluorescent

Agonist-induced endocytosis and processing of the G protein–coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

scholarly journals Mutations in the D1 Domain Inhibit Propeptide-Dependent Multimerization, Trafficking and Regulated Secretion of Von Willebrand Factor in VWD

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1495-1495
Author(s):  
Jie Yin ◽  
Zhenni Ma ◽  
Jian Su ◽  
Xiaojuan Zhao ◽  
Zhaoyue Wang ◽  
...  
Keyword(s):  
Full Length ◽  
Basal Secretion ◽  
Hek 293 ◽  
Transfected Cells ◽  
Regulated Secretion ◽  
293 Cells ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Gene mutations play an important role in the pathogenesis of von Willebrand disease (VWD), resulting in the qualitative defect or quantitative deficit of von Willebrand factor (VWF). VWF propeptide is composed of D1 and D2 domain, which acts as a covalent oxidoreductase in the multimerization. In addition, the propeptide is necessary for the transport from endoplasmic reticulum (ER) to Golgi-apparatus, basal secretion and regulated secretion of VWF. However, the mechanisms of the mutation in D1 domain impairing VWF multimerization and causing low VWF levels in patients remain unknown. Herein we identified four mutations in the D1 domain from VWD patients, and assessed the effect of these mutations on the function of propeptide. We identified p.G39R, p.D141N, p.K157E, and p.C379G in three VWD patients. To characterize the roles of four mutations in propeptide-depend multimerization, we generated several truncated-VWFs, D1D2D¡¯D3 (residues 1-1241), including G39R, K157E, D141N, C379G and wide type (WT). These constructs were then expressed in HEK 293 cells, and were evaluated the D¡¯D3-dimer formation of mutations and WT. Full-length VWF comprising mutations and WT were also restructured and were transfected in HEK 293 cells. We then analyzed the VWF multimer distribution and VWF antigen in the cell supernatant and cell lysate. We also assessed VWF retention in ER and the stimulation secretion by phorbol-12-myristate-13- acetate (PMA) of mutations and those of WT. Two type 3 and 1 type 1 patients were enrolled in our study. VWF antigen were 3, 1 and 8 IU/dL, and VWF:Rco were 2.1, 2.3 and 5.6 IU/dL respectively in three patients. VWF multimer distribution exhibited none in two type 3 patients and normal-like multimer pattern in the type 1. Sequence analysis of VWF gene showed two heterozygous mutations (p.G39R and D141N) in D1 domain of one type 3 patient, and a heterozygous K157E in propeptide and a heterozygous C1165R in D3 domain of the other patient with type 3. Type 1 patients had a heterozygous C379G mutation. Among these four mutations in D1 domain, p.G39R, p.K157E, and p.C379G were novel. In the supernatant of transfected cell, dimerization of D¡¯D3 was absent in truncated-G39R. Compared with that of truncated-WT, decreased but detectable dimerizations were detected in K157E, D141N, and C379G. Similar results were also observed in multimerization of full-length constructs. The multimer assembly was too low to visualize in G39R, whereas decreased medium and absent large VWF multimers were seen in K157E, D141N, and C379G. By immunofluorescence imaging, all full-length VWF variants were detained in ER in different degrees (fig 1). The basal secretions of G39R, K157E, D141N, and C379G were (2.7¡À0. 3)%, (2.5¡À0.2)%, (26.0¡À4.1)%, and (22.4¡À3.8)% of WT respectively. However, VWF antigens in the lysate of transfected cells were (116.5¡À5.4)%, (90.9¡À3.0)%, (91.7¡À0.4)% and (113.8¡À2.9)% of WT for G39R, K157E, D141N, and C379G. No detectable secretion increases of mutant VWF induced by PMA were observed in the transfected cells, while WT-VWF with PMA exhibited increased secretion from 0.69% to 1.66%. These four mutations in D1 domain downgraded the activity of propeptide as the covalent oxidoreductase, and impaired the muiltimeriztion induced by propeptide. They also interfered with VWF transport from ER to Golgi-apparatus and caused the VWF retentions in ER. Therefore, they further reduced the basal secretion and regulated secretion of mature VWF, which could explain the possible pathogenesis of quantitative deficit of VWF in VWD. Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3236-3245 ◽  
Author(s):  
E. Filardo ◽  
J. Quinn ◽  
Y. Pang ◽  
C. Graeber ◽  
S. Shaw ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Intracellular Camp ◽  
Calcium Stores ◽  
G Protein Coupled Receptor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
G Protein Coupled

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17β-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17β-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

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.

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.

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