RESUMO
The SLC4A1/AE1 gene encodes the electroneutral Cl(-)/HCO(3)(-) exchanger of erythrocytes and renal type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl(-)/HCO(3)(-) exchange or (36)Cl(-) efflux but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO(4)(2-)(i)/SO(4)(2-)(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO(4)(2-)(i)/Cl(-)(o) exchange). Whereas wild-type AE1 mediates 1:1 H(+)/SO(4)(2-) cotransport in exchange for either Cl(-) or for the H(+)/SO(4)(2-) ion pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wild-type AE1-mediated SO(4)(2-) transport, both SO(4)(2-)(i)/Cl(-)(o) exchange and SO(4)(2-)(i)/SO(4)(2-)(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pH(o) and activated by alkaline pH(o). hAE1 E681OH displays a similarly altered pH(o) dependence of SO(4)(2-)(i)/Cl(-)(o) exchange. Elevated [SO(4)(2-)](i) increases the K(1/2) of Ae1 E699Q for both extracellular Cl(-) and SO(4)(2-), while reducing inhibition of both exchange mechanisms by acid pH(o). The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO(4)(2-). Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide and should continue to provide valuable paths toward understanding substrate selectivity and self-inhibition in SLC4 anion transporters.
Assuntos
Substituição de Aminoácidos , Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Ânions/metabolismo , Sulfatos/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/genética , Boroidretos/química , Cloretos/metabolismo , Feminino , Ácido Glutâmico/química , Humanos , Concentração de Íons de Hidrogênio , Isoxazóis/química , Cinética , Mesilatos/metabolismo , Camundongos , Oócitos/metabolismo , Ácidos Sulfônicos/metabolismo , Xenopus laevisRESUMO
The mouse is refractory to lithogenic agents active in rats and humans, and so has been traditionally considered a poor experimental model for nephrolithiasis. However, recent studies have identified slc26a6 as an oxalate nephrolithiasis gene in the mouse. Here we extend our earlier demonstration of different anion selectivities of the orthologous mouse and human SLC26A6 polypeptides to investigate the correlation between species-specific differences in SLC26A6 oxalate/anion exchange properties as expressed in Xenopus oocytes and in reported nephrolithiasis susceptibility. We find that human SLC26A6 mediates minimal rates of Cl(-) exchange for Cl(-), sulphate or formate, but rates of oxalate/Cl(-) exchange roughly equivalent to those of mouse slc2a6. Both transporters exhibit highly cooperative dependence of oxalate efflux rate on extracellular [Cl(-)], but whereas the K(1/2) for extracellular [Cl(-)] is only 8 mM for mouse slc26a6, that for human SLC26A6 is 62 mM. This latter value approximates the reported mean luminal [Cl(-)] of postprandial human jejunal chyme, and reflects contributions from both transmembrane and C-terminal cytoplasmic domains of human SLC26A6. Human SLC26A6 variant V185M exhibits altered [Cl(-)] dependence and reduced rates of oxalate/Cl(-) exchange. Whereas mouse slc26a6 mediates bidirectional electrogenic oxalate/Cl(-) exchange, human SLC26A6-mediated oxalate transport appears to be electroneutral. We hypothesize that the low extracellular Cl(-) affinity and apparent electroneutrality of oxalate efflux characterizing human SLC26A6 may partially explain the high human susceptibility to nephrolithiasis relative to that of mouse. SLC26A6 sequence variant(s) are candidate risk modifiers for nephrolithiasis.
Assuntos
Antiporters/genética , Cloretos/metabolismo , Predisposição Genética para Doença/genética , Proteínas de Membrana Transportadoras/genética , Nefrolitíase/genética , Nefrolitíase/metabolismo , Oxalatos/metabolismo , Animais , Antiporters/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Hiperoxalúria/metabolismo , Potenciais da Membrana , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Especificidade da Espécie , Transportadores de Sulfato , Transfecção , Xenopus laevisRESUMO
The SLC26 anion transporter polypeptides exhibit considerably greater sequence diversity among near-species orthologues than is found among the SLC4 bicarbonate transporters, and among SLC26 transporters is most marked among SLC26A6 orthologues. This observation prompted systematic functional comparison in Xenopus oocytes of mouse Slc26a6 with several human SLC26A6 polypeptide variants. Mouse and human polypeptides exhibited similar rates of bidirectional [14C]oxalate flux, Cl-/HCO3- exchange, and Cl-/OH- exchange, and similar cAMP-stimulation and enhancement of that stimulation by wild-type but not delta F508 CFTR. However, high rates of 36Cl- and 35S-sulfate transport by mouse Slc26a6 contrasted with low transport rates of the human proteins. The high 36Cl- transport phenotype cosegregated with the transmembrane domain of mouse Slc26a6 in chimera studies. Mouse Slc26a6 and human SLC26A6 each mediated electroneutral Cl-/HCO3- and Cl-/OH- exchange. But, whereas Cl-/oxalate exchange by mouse Slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. Oocyte expression of either mouse or human orthologue elicited currents that were pharmacologically distinct from the monovalent anion exchange activities measured in the same lots of oocytes. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive in isotopic flux assays. Understanding of SLC26 transport mechanisms and pathophysiology will benefit from recognition of substantial differences in transport properties among orthologues.
Assuntos
Antiporters/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Animais , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Humanos , Camundongos , Oxalatos/metabolismo , Estrutura Terciária de Proteína , Transportadores de Sulfato , Sulfatos/metabolismoRESUMO
The mouse anion exchanger AE2/SLC4A2 Cl(-)/HCO(-)(3) exchanger is essential to post-weaning life. AE2 polypeptides regulate pH(i), chloride concentration, cell volume, and transepithelial ion transport in many tissues. Although the AE2a isoform has been extensively studied, the function and regulation of the other AE2 N-terminal variant mRNAs of mouse (AE2b1, AE2b2, AE2c1, and AE2c2) have not been examined. We now present an extended analysis of AE2 variant mRNA tissue distribution and function. We show in Xenopus oocytes that all AE2 variant polypeptides except AE2c2 mediated Cl(-) transport are subject to inhibition by acidic pH(i) and to activation by hypertonicity and NH(+)(4). However, AE2c1 differs from AE2a, AE2b1, and AE2b2 in its alkaline-shifted pH(o)((50)) (7.70 +/- 0.11 versus 6.80 +/- 0.05), suggesting the presence of a novel AE2a pH-sensitive regulatory site between amino acids 99 and 198. Initial N-terminal deletion mutagenesis restricted this site to the region between amino acids 120 and 150. Further analysis identified AE2a residues 127-129, 130-134, and 145-149 as jointly responsible for the difference in pH(o)((50)) between AE2c1 and the longer AE2a, AE2b1, and AE2b2 polypeptides. Thus, AE2c1 exhibits a unique pH(o) sensitivity among the murine AE2 variant polypeptides, in addition to a unique tissue distribution. Physiological coexpression of AE2c1 with other AE2 variant polypeptides in the same cell should extend the range over which changing pH(o) can regulate AE2 transport activity.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/fisiologia , Antiporters/genética , Antiporters/fisiologia , Animais , Proteínas de Transporte de Ânions/química , Antiporters/química , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato , Cromatografia por Troca Iônica/métodos , Citoplasma/metabolismo , DNA Complementar/metabolismo , Deleção de Genes , Variação Genética , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Oócitos/metabolismo , Peptídeos/química , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SLC4A , Distribuição Tecidual , Transcrição Gênica , XenopusRESUMO
The shark liver antimicrobial polyaminosterol squalamine is an angiogenesis inhibitor under clinical investigation as an anti-cancer agent and as a treatment for the choroidal neovascularization associated with macular degeneration of the retina. The related polyaminosterol MSI-1436 is an appetite suppressant that decreases systemic insulin resistance. However, the mechanisms of action of these polyaminosterols are unknown. We report effects of MSI-1436 on Xenopus oocytes consistent with the existence of a receptor for polyaminosterols. MSI-1436 activates bidirectional, trans-chloride-independent Cl- flux in Xenopus oocytes. At least part of this DIDS-sensitive Cl- flux is conductive, as measured using two-electrode voltage-clamp and on-cell patch-clamp techniques. MSI-1436 also elevates cytosolic Ca2+ concentration ([Ca2+]) and increases bidirectional 45Ca2+ flux. Activation of Cl- flux and elevation of cytosolic [Ca2+] by MSI-1436 both are accelerated by lowering bath Ca2+ and are not acutely inhibited by extracellular EGTA. Elevation of cytosolic [Ca2+] by MSI-1436 requires heparin-sensitive intracellular Ca2+ stores. Although injected EGTA abolishes the increased conductive Cl- flux, that Cl- flux is not dependent on heparin-sensitive stores. In low-bath Ca2+ conditions, several structurally related polyaminosterols act as strong agonists or weak agonists of conductive Cl- flux in oocytes. Weak agonist polyaminosterols antagonize the strong agonist, MSI-1436, but upon addition of the conductive Cl- transport inhibitor DIDS, they are converted into strong agonists. Together, these properties operationally define a polyaminosterol receptor at or near the surface of the Xenopus oocyte, provide an initial description of receptor signaling, and suggest routes toward further understanding of a novel class of appetite suppressants and angiogenesis inhibitors.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/fisiologia , Cloro/metabolismo , Oócitos/fisiologia , Receptores de Superfície Celular/metabolismo , Tubarões/metabolismo , Esteróis/farmacologia , Animais , Fatores Biológicos/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Células Cultivadas , Canais de Cloreto/efeitos dos fármacos , Colestanóis , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Fígado/química , Xenopus laevisRESUMO
Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl(-) channel activity in Xenopus oocytes. Background. Cyst expansion in autosomal-dominant polycystic kidney disease (ADPKD) is characterized by active Cl(-) secretion in excess of solute reabsorption. However, the connections between elevated epithelial Cl(-) secretion and loss-of-function or dysregulation of either ADPKD gene polycystin-1 (PC1) or polycystin-2 (PC2) remain little understood. Methods. Cl(-) transport in Xenopus oocytes expressing the CD16.7-PKD1 (115-226) fusion protein containing the final 112 amino acid (aa) of the PC1 C-terminal cytoplasmic tail, or in oocytes expressing related PC1 fusion protein mutants, was studied by isotopic flux, two-electrode voltage clamp, and outside-out patch clamp recording. Results. Expression in oocytes of CD16.7-PKD1 (115-226) increased rates of both influx and efflux of (36)Cl(-), whereas CD16.7-PKD1 (1-92) containing the initial 92 aa of the PC1 C-terminal cytoplasmic tail was inactive. The increased Cl(-) transport resembled CD16.7-PKD1 (115-226)-stimulated cation current in its sensitivity to ADPKD-associated missense mutations, to mutations in phosphorylation sites, and to mutations within or encroaching upon the PC1 coiled-coil domain, as well as in its partial suppression by coexpressed PC2. The NS3623- and 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive (36)Cl(-) flux was not blocked by injected ethyleneglycol tetraacetate (EGTA) or by the cation channel inhibitor SKF96365, and was stimulated by the cation channel inhibitor La(3+), suggesting that CD16.7-PKD1 (115-226)-associated cation conductance was not required for (36)CI(-) flux activation. Outside-out patches from oocytes expressing CD16.7-PKD1 (115-226) also exhibited increased NS3623-sensitive Cl(-) current. Conclusion. These data show that CD16.7-PKD1 (115-226) activates Cl(-) channels in the Xenopus oocyte plasma membrane in parallel with, but not secondary to, activation of Ca(2+)-permeable cation channels.
Assuntos
Canais de Cloreto/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas/genética , Proteínas/metabolismo , Animais , Cálcio/metabolismo , Cátions/metabolismo , Membrana Celular/metabolismo , Cloretos/metabolismo , Citoplasma/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Oócitos/citologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Fosforilação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/fisiopatologia , Estrutura Terciária de Proteína , Proteínas/química , RNA Complementar , Radioisótopos , Serina/metabolismo , Canais de Cátion TRPP , Tirosina/metabolismo , XenopusRESUMO
The slc4 and slc26 gene families encode two distinct groups of gene products that transport HCO(3)(-) 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(-)/HCO(3)(-) 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.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Transporte de Ânions/metabolismo , Caenorhabditis elegans/metabolismo , Clonagem Molecular , DNA Complementar , Expressão Gênica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência , TransgenesRESUMO
The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.
Assuntos
Antiporters/genética , Antiporters/fisiologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Animais , Ânions , Transporte Biológico , Cloro/química , Cromatografia por Troca Iônica , Códon , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Eletrodos , Variação Genética , Humanos , Concentração de Íons de Hidrogênio , Immunoblotting , Camundongos , Microscopia Confocal , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Oócitos/metabolismo , Oxalatos/metabolismo , Peptídeos/química , Fenótipo , Estrutura Terciária de Proteína , RNA Complementar/metabolismo , Bicarbonato de Sódio/química , Espectrometria de Fluorescência , Transportadores de Sulfato , Sulfatos/química , Fatores de Tempo , XenopusRESUMO
We have previously defined in the NH2-terminal cytoplasmic domain of the mouse AE2/SLC4A2 anion exchanger a critical role for the highly conserved amino acids (aa) 336-347 in determining wild-type pH sensitivity of anion transport. We have now engineered hexa-Ala ((A)6) and individual amino acid substitutions to investigate the importance to pH-dependent regulation of AE2 activity of the larger surrounding region of aa 312-578. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during changes in pHi or pHo in HEPES-buffered and in 5% CO2/HCO3- -buffered conditions. Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited at low pHo, with a pHo(50) value = 6.75 +/- 0.05 and was stimulated up to 10-fold by intracellular alkalinization. Individual mutation of several amino acid residues at non-contiguous sites preceding or following the conserved sequence aa 336-347 attenuated pHi and/or pHo sensitivity of 36Cl- efflux. The largest attenuation of pH sensitivity occurred with the AE2 mutant (A)6357-362. This effect was phenocopied by AE2 H360E, suggesting a crucial role for His360. Homology modeling of the three-dimensional structure of the AE2 NH2-terminal cytoplasmic domain (based on the structure of the corresponding region of human AE1) predicts that those residues shown by mutagenesis to be functionally important define at least one localized surface region necessary for regulation of AE2 activity by pH.
Assuntos
Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/metabolismo , Antiporters/química , Antiporters/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Antiportadores de Cloreto-Bicarbonato , Cloretos/metabolismo , Sequência Conservada , Feminino , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Transporte de Íons , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas SLC4A , Homologia de Sequência de Aminoácidos , Xenopus , Domínios de Homologia de srcRESUMO
Cl-/HCO3- exchange activity mediated by the AE1 anion exchanger is reduced by carbonic anhydrase II (CA2) inhibition or by prevention of CA2 binding to the AE1 C-terminal cytoplasmic tail. This type of AE1 inhibition is thought to represent reduced metabolic channeling of HCO3- to the intracellular HCO3- binding site of AE1. To test the hypothesis that CA2 binding might itself allosterically activate AE1 in Xenopus oocytes, we compared Cl-/Cl- and Cl-/HCO3- exchange activities of AE1 polypeptides with truncation and missense mutations in the C-terminal tail. The distal renal tubular acidosis-associated AE1 901X mutant exhibited both Cl-/Cl- and Cl-/HCO3- exchange activities. In contrast, AE1 896X, 891X, and AE1 missense mutants in the CA2 binding site were inactive as Cl-/HCO3- exchangers despite exhibiting normal Cl-/Cl- exchange activities. Co-expression of CA2 enhanced wild-type AE1-mediated Cl-/HCO3- exchange, but not Cl-/Cl- exchange. CA2 co-expression could not rescue Cl-/HCO3- exchange activity in AE1 mutants selectively impaired in Cl-/HCO3- exchange. However, co-expression of transport-incompetent AE1 mutants with intact CA2 binding sites completely rescued Cl-/HCO3- exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl-/HCO3- exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO3- transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Bicarbonatos/metabolismo , Anidrase Carbônica II/metabolismo , Cloretos/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/genética , Ânions , Sítios de Ligação/genética , Transporte Biológico , Anidrase Carbônica II/genética , Dimerização , Feminino , Deleção de Genes , Expressão Gênica , Humanos , Bicamadas Lipídicas/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese , Mutação de Sentido Incorreto , Oócitos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Relação Estrutura-Atividade , Transfecção , XenopusRESUMO
Three different types of cation exchangers were produced from four basic poly(styrene-divinylbenzene) substrates with different properties. Porous PS-DVB resin beads were functionalized by sulfonation and sulfoacylation under various conditions to produce sulfonated resins with exchange capacities of 0.03-1.80 mM g(-1). The matrix with 50% of cross-linking is most suitable for updating by the proposed technique. Sulfuric and chlorosulfonic acids were used as the reagents for sulfonation. The sulfonating conditions, capacities, and the technique of the synthesis are given. The effects of parameters of the sulfonation reaction and the composition of the reactionary mixture on ion-exchange capacity of the sorbents were investigated. Selectivity and efficiency of the separation of some inorganic cations and derivatives of amines and hydrazines on the resins obtained are compared by ion chromatography with conductometric detection. As a result, the sulfoacylated resin was proved more efficient for the separation of these analytes. The resolution of the analytes strongly depends on the degree of functionalization. The best performance about 19,000 plates m(-1) was obtained using the sulfopropionylated beads with an ion-exchange capacity of 0.3 mM g(-1). The prepared sulfoacylated cation exchanger was compared with the commercially available Dionex CS-12 packing material. It was found that the separation of alkaline ions and 1,1-dimethylhydrazine (UDMH) were much better that those for the commercial material.
Assuntos
Aminas/análise , Resinas de Troca de Cátion , Cromatografia por Troca Iônica/instrumentação , Cromatografia por Troca Iônica/métodos , Hidrazinas/análise , Poliestirenos , Compostos de Vinila , Alcanossulfonatos , Aminas/química , Análise de Falha de Equipamento , Hidrazinas/química , Teste de Materiais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Eritropoese/genética , Mitose/genética , Mutação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Anemia Diseritropoética Congênita/genética , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Peixe-Zebra/sangueRESUMO
Mutations in the human SLC26A3 gene, also known as down-regulated in adenoma (hDRA), cause autosomal recessive congenital chloride-losing diarrhoea (CLD). hDRA expressed in Xenopus oocytes mediated bidirectional Cl--Cl- and Cl--HCO3- exchange. In contrast, transport of oxalate was low, and transport of sulfate and of butyrate was undetectable. Two CLD missense disease mutants of hDRA were nonfunctional in oocytes. Truncation of up to 44 C-terminal amino acids from the putatively cytoplasmic C-terminal hydrophilic domain left transport function unimpaired, but deletion of the adjacent STAS (sulfate transporter anti-sigma factor antagonist) domain abolished function. hDRA-mediated Cl- transport was insensitive to changing extracellular pH, but was inhibited by intracellular acidification and activated by NH4+ at acidifying concentrations. These regulatory responses did not require the presence of either hDRA's N-terminal cytoplasmic tail or its 44 C-terminal amino acids, but they did require more proximate residues of the C-terminal cytoplasmic domain. Although only weakly sensitive to inhibition by stilbenes, hDRA was inhibited with two orders of magnitude greater potency by the anti-inflammatory drugs niflumate and tenidap. cAMP-insensitive Cl--HCO3- exchange mediated by hDRA gained modest cAMP sensitivity when co-expressed with cystic fibrosis transmembrane conductance regulator (CFTR). Despite the absence of hDRA transcripts in human cell lines derived from CFTR patients, DRA mRNA was present at wild-type levels in proximal colon and nearly so in the distal ileum of CFTR(-/-) mice. Thus, pharmacological modulation of DRA might be a useful adjunct treatment of cystic fibrosis.
Assuntos
Antiporters , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Animais , Bicarbonatos/metabolismo , Proteínas de Transporte/química , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato , Cloretos/metabolismo , Colo/fisiologia , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citoplasma , Diarreia/congênito , Diarreia/genética , Diarreia/fisiopatologia , Feminino , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Soluções Hipertônicas/farmacologia , Íleo/fisiologia , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos CFTR , Mutagênese , Mutação de Sentido Incorreto , Oócitos/fisiologia , Estrutura Terciária de Proteína , Transportadores de Sulfato , XenopusRESUMO
We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH (pH(i)) and extracellular pH (pH(o)). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pH(i): an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH(4)(+). We find that both NH(2)-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the NH(2)-terminal cytoplasmic domain, an alanine scan of AE2 amino acids 336-347 identified residues whose individual mutation abolished or severely attenuated sensitivity to both or only one activating stimulus. Chelation of cytoplasmic Ca(2+) (Ca(i)(2+)) diminished or abolished AE2 stimulation by NH(4)(+) and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of Ca(2+) signaling. Unlike AE2 stimulation by NH(4)(+) and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain.
Assuntos
Proteínas de Transporte de Ânions , Antiporters , Cálcio/metabolismo , Citoplasma/metabolismo , Ácido Egtázico/análogos & derivados , Soluções Hipertônicas/farmacologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Compostos de Amônio Quaternário/farmacologia , Animais , Quelantes/farmacologia , Ácido Egtázico/farmacologia , Feminino , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Proteínas de Membrana/genética , Mutação/fisiologia , Oócitos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Proteínas SLC4A , Relação Estrutura-Atividade , XenopusRESUMO
Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na+-independent chloride-bicarbonate exchanger genes and multiple Na+-bicarbonate cotransporter and Na+-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na+-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.
Assuntos
Proteínas de Transporte de Ânions , Antiporters , Antiportadores de Cloreto-Bicarbonato/química , Concentração de Íons de Hidrogênio , Proteínas de Membrana/química , Proteínas de Membrana/genética , Animais , Ânions , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/fisiologia , Citoplasma/química , Regulação da Expressão Gênica , Humanos , Família Multigênica , Mutagênese , Compostos de Amônio Quaternário/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas SLC4A , Relação Estrutura-AtividadeRESUMO
Tubular acid-base transport regulates systemic acid-base balance. Transepithelial acid-base transport across nephron segments requires the coordinated control of intracellular pH and cellular volume by transporters of protons and bicarbonate. Bicarbonate transporter polypeptides are encoded by at least two gene families, SLC4 and SLC26. The SLC4 gene family includes at least three Na()+)-independent chloride-bicarbonate exchanger genes and multiple Na(+)-bicarbonate cotransporter and Na(+)-dependent anion exchanger genes. The most extensively studied among them are the Na(+)-independent anion exchangers, AE1, AE2, and AE3, all of which are expressed in kidney. The AE1 gene encodes eAE1 (band 3), the major intrinsic protein of the erythrocyte, as well as kAE1, the basolateral Cl/HCO3 exchanger of the acid-secreting Type A intercalated cell. Mutations in AE1 are responsible for some forms of heritable distal renal tubular acidosis. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by ammonium ion. These properties are not shared by the closely related AE1 anion exchanger. Less is known about AE3 in kidney. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2 which are important in exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.
Assuntos
Proteínas de Transporte de Ânions , Antiportadores de Cloreto-Bicarbonato/genética , Família Multigênica , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Antiporters/genética , Antiporters/fisiologia , Antiportadores de Cloreto-Bicarbonato/química , Antiportadores de Cloreto-Bicarbonato/fisiologia , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Estrutura Secundária de Proteína , Proteínas SLC4A , Relação Estrutura-AtividadeRESUMO
Polycystin-1 (PKD1) mutations account for approximately 85% of autosomal dominant polycystic kidney disease (ADPKD). We have shown previously that oocyte surface expression of a transmembrane fusion protein encoding part of the cytoplasmic COOH terminus of PKD1 increases activity of a Ca2+-permeable cation channel. We show here that human ADPKD mutations incorporated into this fusion protein attenuated or abolished encoded cation currents. Point mutations and truncations showed that cation current expression requires integrity of a region encompassing the putative coiled coil domain of the PKD1 cytoplasmic tail. Whereas these loss-of-function mutants did not exhibit dominant negative phenotypes, coexpression of a fusion protein expressing the interacting COOH-terminal cytoplasmic tail of PKD2 did suppress cation current. Liganding of the ectodomain of the PKD1 fusion protein moderately activated cation current. The divalent cation permeability and pharmacological profile of the current has been extended. Inducible expression of the PKD1 fusion in EcR-293 cells was also associated with activation of cation channels and increased Ca2+ entry.