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1.
PLoS One ; 19(1): e0296928, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38252645

RESUMO

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.


Assuntos
Proteínas de Transporte de Ânions , Antiporters , Distrofias Hereditárias da Córnea , Distrofia Endotelial de Fuchs , Humanos , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Transtornos Cromossômicos , Distrofias Hereditárias da Córnea/genética , Células Endoteliais , Distrofia Endotelial de Fuchs/genética , Mutação , Proteínas SLC4A
2.
Nat Commun ; 15(1): 759, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38272905

RESUMO

Anion exchanger 2 (AE2) is an electroneutral Na+-independent Cl-/HCO3- exchanger belongs to the SLC4 transporter family. The widely expressed AE2 participates in a variety of physiological processes, including transepithelial acid-base secretion and osteoclastogenesis. Both the transmembrane domains (TMDs) and the N-terminal cytoplasmic domain (NTD) are involved in regulation of AE2 activity. However, the regulatory mechanism remains unclear. Here, we report a 3.2 Å cryo-EM structure of the AE2 TMDs in complex with PIP2 and a 3.3 Å full-length mutant AE2 structure in the resting state without PIP2. We demonstrate that PIP2 at the TMD dimer interface is involved in the substrate exchange process. Mutation in the PIP2 binding site leads to the displacement of TM7 and further stabilizes the interaction between the TMD and the NTD. Reduced substrate transport activity and conformation similar to AE2 in acidic pH indicating the central contribution of PIP2 to the function of AE2.


Assuntos
Antiporters , Lipídeos , Humanos , Antiportadores de Cloreto-Bicarbonato/genética , Antiporters/genética , Proteínas SLC4A , Mutação , Proteínas de Transporte de Ânions/metabolismo , Concentração de Íons de Hidrogênio
3.
Sheng Li Xue Bao ; 75(1): 137-150, 2023 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-36859843

RESUMO

The solute carrier family 4 (SLC4) includes 10 members (SLC4A1-5, SLC4A7-11), which are expressed in multiple tissues in the human body. The SLC4 family members differ in their substrate dependence, charge transport stoichiometry and tissue expression. Their common function is responsible for the transmembrane exchange of multiple ions, which is involved in many important physiological processes, such as erythrocyte CO2 transport and the regulation of cell volume and intracellular pH. In recent years, many studies have focused on the role of SLC4 family members in the occurrence of human diseases. When SLC4 family members have gene mutations, a series of functional disorders will occur in the body, leading to the occurrence of some diseases. This review summarizes the recent progress about the structures, functions and disease correlation of SLC4 members, in order to provide clues for the prevention and treatment of related human diseases.


Assuntos
Mutação , Proteínas SLC4A , Humanos , Proteínas SLC4A/genética , Proteínas SLC4A/fisiologia
4.
Br J Ophthalmol ; 106(2): 281-287, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-33879471

RESUMO

BACKGROUND: Autosomal recessive congenital hereditary corneal dystrophy (CHED) is a rare isolated developmental anomaly of the eye characterised by diffuse bilateral corneal clouding that may lead to visual impairment requiring corneal transplantation. CHED is known to be caused by mutations in the solute carrier family 4 member 11 (SLC4A11) gene which encodes a membrane transporter protein (sodium bicarbonate transporter-like solute carrier family 4 member 11). METHODS: To identify SLC4A11 gene mutations associated with CHED (OMIM: #217700), genomic DNA was extracted from whole blood and sequenced for all exons and intron-exon boundaries in two large Tunisian families. RESULTS: A novel deletion SLC4A11 mutation (p. Leu479del; c.1434_1436del) is responsible for CHED in both analysed families. This non-frameshift mutation was found in a homozygous state in affected members and heterozygous in non-affected members. In silico analysis largely support the pathogenicity of this alteration that may leads to stromal oedema by disrupting the osmolarity balance. Being localised to a region of alpha-helical secondary structure, Leu479 deletion may induce protein-compromising structural rearrangements. CONCLUSION: To the best of our knowledge, this is the first clinical and genetic study exploring CHED in Tunisia. The present work also expands the list of pathogenic genotypes in SLC4A11 gene and its associated clinical diagnosis giving more insights into genotype-phenotype correlations.


Assuntos
Proteínas de Transporte de Ânions , Distrofias Hereditárias da Córnea , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Consanguinidade , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/genética , Humanos , Mutação , Proteínas SLC4A/genética
5.
Invest Ophthalmol Vis Sci ; 61(8): 39, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32721020

RESUMO

Purpose: To elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4. Methods: Comparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and Slc4a11 (Slc4a11-/- MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations. Results: CTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK-p53/ULK1 in both SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3- transport activity and altered NH4Cl-induced membrane potential changes were observed in Slc4a11-/- MCEnC. Conclusions: Reduced steady-state ATP levels and subsequent activation of the AMPK-p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.


Assuntos
Trifosfato de Adenosina/biossíntese , Endotélio Corneano , Transporte de Íons/fisiologia , Mitocôndrias/metabolismo , Proteínas SLC4A/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Células Cultivadas , Distrofias Hereditárias da Córnea/genética , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Endotélio Corneano/fisiopatologia , Metabolismo Energético , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutação , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
6.
Int J Mol Sci ; 21(11)2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517126

RESUMO

Bicarbonate uptake is one of the early steps of capacitation, but the identification of proteins regulating anion fluxes remains elusive. The aim of this study is to investigate the role of sperm solute carrier 4 (SLC4) A1 (spAE1) in the capacitation process. The expression, location, and tyrosine-phosphorylation (Tyr-P) level of spAE1 were assessed. Thereby, it was found that 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an SLC4 family channel blocker, inhibited capacitation in a dose-dependent manner by decreasing acrosome reaction (ARC% 24.5 ± 3.3 vs 64.9 ± 4.3, p < 0.05) and increasing the percentage of not viable cells (NVC%), comparable to the inhibition by I-172, a cystic fibrosis transmembrane conductance regulator (CFTR) blocker (AR% 30.5 ± 4.4 and NVC% 18.6 ± 2.2). When used in combination, a synergistic inhibitory effect was observed with a remarkable increase of the percentage of NVC (45.3 ± 4.1, p < 0.001). spAE1 was identified in sperm membrane as a substrate for Tyr-protein kinases Lyn and Syk, which were identified as both soluble and membrane-bound pools. spAE1-Tyr-P level increased in the apical region of sperm under capacitating conditions and was negatively affected by I-172 or DIDS, and, to a far greater extent, by a combination of both. In conclusion, we demonstrated that spAE1 is expressed in sperm membranes and it is phosphorylated by Syk, but above all by Lyn on Tyr359, which are involved in sperm viability and capacitation.


Assuntos
Proteínas SLC4A/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/fisiologia , Tirosina/metabolismo , Reação Acrossômica , Membrana Celular , Sobrevivência Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Masculino , Fosforilação , Proteínas SLC4A/genética
7.
Redox Biol ; 26: 101260, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31254733

RESUMO

SLC4A11 is a NH3 sensitive membrane transporter with H+ channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH3-sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, Slc4a11-/- (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH3 production by GLS1 inhibition or dimethyl αKetoglutarate supplementation, or by BAM15 mitochondrial uncoupling. Slc4a11 KO mouse corneal edema can be partially reversed by αKetoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.


Assuntos
Amônia/metabolismo , Glutamina/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Proteínas SLC4A/metabolismo , Amônia/farmacologia , Animais , Células Endoteliais , Endotélio Corneano/metabolismo , Técnicas de Inativação de Genes , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Modelos Biológicos , Fosforilação Oxidativa , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/metabolismo , Proteínas SLC4A/genética
8.
Nat Commun ; 10(1): 2032, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048734

RESUMO

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.


Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Transportadores de Sulfato/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Deinococcus , Espectroscopia de Ressonância de Spin Eletrônica , Mutagênese Sítio-Dirigida , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas SLC4A/química , Proteínas SLC4A/metabolismo , Transportadores de Sulfato/química , Transportadores de Sulfato/genética , Transportadores de Sulfato/isolamento & purificação
9.
G Ital Nefrol ; 36(1)2019 Feb.
Artigo em Italiano | MEDLINE | ID: mdl-30758150

RESUMO

We describe the case of a 5-year-old who came to our attention for a growth delay. Among the investigations planned because of the child's short stature, we performed an abdominal ultrasound showing normal-sized kidneys with signs of cortico-medullar de-differentiation, diffuse medullary hyperechogenicity with reduction of cortical thickness and cortical-medullary cysts. The ultrasound findings, also confirmed in MRI, led us to suspect a genetically determined cystic nephropathy of the nephronophthisis or medullary cystic disease type. No mutation was identified in NPHP1, HNFb1 and UMOD genes. Interestingly, laboratory investigations revealed a severe metabolic acidosis with normal renal function and hypokalemia. These findings are not characteristics of a nephronophthisis. We therefore also performed molecular analysis for distal tubular acidosis (dRTA) that showed the association of two genetic variants of ATP6V1B1 and SLC4A genes. These "double mutations" have been inherited from the mother, which however does not have the classic dRTA phenotype. These variants do not currently meet the criteria for a conclusive molecular diagnosis of dRTA but represent variants of uncertain clinical significance. However, considering the clinical and laboratory data one can reasonably conclude that the child has a "probable" diagnosis of distal tubular acidosis. The rapid recovery of staturo-ponderal growth after the start of alkalizing treatment supports our diagnostic hypothesis. The association between distal tubular acidosis and renal cysts is well described in the literature. The hypothesis is that chronic hypokalemia may play a possible role in the formation of renal cysts.


Assuntos
Acidose Tubular Renal/genética , Transtornos do Crescimento/complicações , Doenças Renais Císticas/complicações , Proteínas SLC4A/genética , ATPases Vacuolares Próton-Translocadoras/genética , Acidose/diagnóstico , Acidose Tubular Renal/complicações , Acidose Tubular Renal/diagnóstico , Pré-Escolar , Humanos , Hipopotassemia/diagnóstico , Rim/diagnóstico por imagem , Doenças Renais Císticas/diagnóstico por imagem , Masculino , Mutação , Ultrassonografia
10.
Am J Physiol Cell Physiol ; 314(5): C569-C588, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29384695

RESUMO

The SLC4 family Cl-/[Formula: see text] cotransporters (NBCe1, NBCe2, NBCn1, and NBCn2) contribute to a variety of vital physiological processes including pH regulation and epithelial fluid secretion. Accordingly, their dysfunction can have devastating effects. Disorders such as epilepsy, hemolytic anemia, glaucoma, hearing loss, osteopetrosis, and renal tubular acidosis are all genetically linked to SLC4-family gene loci. This review summarizes how studies of Slc4-modified mice have enhanced our understanding of the etiology of SLC4-linked pathologies and the interpretation of genetic linkage studies. The review also surveys the novel disease signs exhibited by Slc4-modified mice which could either be considered to presage their description in humans, or to highlight interspecific differences. Finally, novel Slc4-modified mouse models are proposed, the study of which may further our understanding of the basis and treatment of SLC4-linked disorders of [Formula: see text]-transporter dysfunction.


Assuntos
Equilíbrio Ácido-Base/genética , Bicarbonatos/metabolismo , Túbulos Renais/metabolismo , Proteínas SLC4A/genética , Proteínas SLC4A/metabolismo , Animais , Modelos Animais de Doenças , Predisposição Genética para Doença , Concentração de Íons de Hidrogênio , Túbulos Renais/fisiopatologia , Camundongos Transgênicos , Fenótipo
11.
EBioMedicine ; 16: 292-301, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28117276

RESUMO

Corneal endothelium (CE) is among the most metabolically active tissues in the body. This elevated metabolic rate helps the CE maintain corneal transparency by its ion and fluid transport properties, which when disrupted, leads to visual impairment. Here we demonstrate that glutamine catabolism (glutaminolysis) through TCA cycle generates a large fraction of the ATP needed to maintain CE function, and this glutaminolysis is severely disrupted in cells deficient in NH3:H+ cotransporter Solute Carrier Family 4 Member 11 (SLC4A11). Considering SLC4A11 mutations leads to corneal endothelial dystrophy and sensorineural deafness, our results indicate that SLC4A11-associated developmental and degenerative disorders result from altered glutamine catabolism. Overall, our results describe an important metabolic mechanism that provides CE cells with the energy required to maintain high level transport activity, reveal a direct link between glutamine metabolism and developmental and degenerative neuronal diseases, and suggest an approach for protecting the CE during ophthalmic surgeries.


Assuntos
Trifosfato de Adenosina/metabolismo , Endotélio Corneano/metabolismo , Metabolismo Energético , Glutamina/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Ciclo do Ácido Cítrico , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/patologia , Expressão Gênica , Humanos , Transporte de Íons , Camundongos Knockout , Microscopia de Fluorescência , Mutação , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SLC4A/genética , Proteínas SLC4A/metabolismo
12.
Hum Mutat ; 35(9): 1082-91, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24916015

RESUMO

SLC4A11 mutations cause some cases of the corneal endothelial dystrophies, congenital hereditary endothelial corneal dystrophy type 2 (CHED2), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). SLC4A11 protein was recently identified as facilitating water flux across membranes. SLC4A11 point mutations usually cause SLC4A11 misfolding and retention in the endoplasmic reticulum (ER). We set about to test the feasibility of rescuing misfolded SLC4A11 protein to the plasma membrane as a therapeutic approach. Using a transfected HEK293 cell model, we measured functional activity present in cells expressing SLC4A11 variants in combinations representing the state found in CHED2 carriers, affected CHED2, FECD individuals, and unaffected individuals. These cells manifest respectively about 60%, 5%, and 25% of the water flux activity, relative to the unaffected (WT alone). ER-retained CHED2 mutant SLC4A11 protein could be rescued to the plasma membrane, where it conferred 25%-30% of WT water flux level. Further, some ER-retained CHED2 mutants expressed at 30°C supported increased water flux compared with 37°C cultures. Caspase activation and cell vitality assays revealed that expression of SLC4A11 mutants in HEK293 cells does not induce cell death. We conclude that therapeutics able to increase cell surface localization of ER-retained SLC4A11 mutants hold promise to treat CHED2 and FECD patients.


Assuntos
Distrofias Hereditárias da Córnea/genética , Mutação , Proteínas SLC4A/genética , Apoptose/genética , Caspase 3/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Retículo Endoplasmático/metabolismo , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico , Deficiências na Proteostase/genética , Proteínas SLC4A/química , Proteínas SLC4A/metabolismo , Temperatura
13.
Pflugers Arch ; 466(8): 1501-16, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24515290

RESUMO

SLC4 transporters are membrane proteins that in general mediate the coupled transport of bicarbonate (carbonate) and share amino acid sequence homology. These proteins differ as to whether they also transport Na(+) and/or Cl(-), in addition to their charge transport stoichiometry, membrane targeting, substrate affinities, developmental expression, regulatory motifs, and protein-protein interactions. These differences account in part for the fact that functionally, SLC4 transporters have various physiological roles in mammals including transepithelial bicarbonate transport, intracellular pH regulation, transport of Na(+) and/or Cl(-), and possibly water. Bicarbonate transport is not unique to the SLC4 family since the structurally unrelated SLC26 family has at least three proteins that mediate anion exchange. The present review focuses on the first of the sodium-dependent SLC4 transporters that was identified whose structure has been most extensively studied: the electrogenic Na(+)-base cotransporter NBCe1. Mutations in NBCe1 cause proximal renal tubular acidosis (pRTA) with neurologic and ophthalmologic extrarenal manifestations. Recent studies have characterized the important structure-function properties of the transporter and how they are perturbed as a result of mutations that cause pRTA. It has become increasingly apparent that the structure of NBCe1 differs in several key features from the SLC4 Cl(-)-HCO3 (-) exchanger AE1 whose structural properties have been well-studied. In this review, the structure-function properties and regulation of NBCe1 will be highlighted, and its role in health and disease will be reviewed in detail.


Assuntos
Acidose Tubular Renal/genética , Antiportadores de Cloreto-Bicarbonato/fisiologia , Proteínas SLC4A/fisiologia , Simportadores de Sódio-Bicarbonato/fisiologia , Acidose Tubular Renal/fisiopatologia , Animais , Transporte Biológico/genética , Humanos , Proteínas de Membrana Transportadoras/fisiologia , Relação Estrutura-Atividade
14.
Hum Mol Genet ; 22(22): 4579-90, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23813972

RESUMO

Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11(-/-) mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.


Assuntos
Distrofias Hereditárias da Córnea/genética , Substância Própria/fisiopatologia , Proteínas SLC4A/metabolismo , Água/metabolismo , Animais , Aquaporina 1/metabolismo , Aquaporinas/metabolismo , Córnea/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Substância Própria/metabolismo , Substância Própria/patologia , Células HEK293 , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Oócitos/metabolismo , Fenótipo , Proteínas SLC4A/genética , Transdução de Sinais/genética , Xenopus laevis
15.
J Cell Physiol ; 228(10): 2042-53, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23553550

RESUMO

The coupled action of the Na(+)/H(+) exchanger NHE1 and the HCO3(-)/Cl(-) exchanger AE2 constitutes the principal mechanism for acute correction of decreased cell volume in mammalian somatic cells, while, when acting separately, they regulate intracellular pH. It was previously found that AE2 becomes inactivated during meiosis in mouse oocytes. Similarly, NHE1 activity stimulated by intracellular acidosis was present in preovulatory germinal vesicle stage (GV) mouse oocytes and then decreased during meiotic maturation. In contrast, NHE1 activity stimulated by decreased cell volume was low in GV oocytes but became active during meiotic maturation as the oocyte detached from the zona pellucida. It then decreased again in mature eggs similar to activity stimulated by acidosis. The subcellular localization of NHE1 was investigated with YFP-tagged NHE1. Exogenous NHE1 expressed in GV oocytes localized to the plasma membrane and resulted in increased Na(+)/H(+) exchanger activity, but only when co-expressed with calcineurin homologous protein 1 (CHP1). When oocytes expressing functional NHE1 were matured to eggs, however, membrane localization of NHE1 and Na(+)/H(+) exchanger activity were lost. It was unknown why NHE1 and AE2 activities are suppressed during meiotic maturation. Maintenance of cell volume in preimplantation embryos requires glycine accumulation via the GLYT1 transporter, a process unique to eggs and early embryos that is initiated during meiotic maturation. When NHE1 and AE2 activities were maintained in GV oocytes by exogenous expression, glycine accumulation was inhibited. We propose that NHE1-mediated acute cell volume regulation is inactivated during meiotic maturation to allow preferential accumulation of glycine in eggs.


Assuntos
Glicina/metabolismo , Meiose/fisiologia , Oócitos/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Antiporters/genética , Antiporters/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Tamanho Celular , Feminino , Glicina/genética , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Concentração de Íons de Hidrogênio , Meiose/genética , Camundongos , Oócitos/metabolismo , Ratos , Proteínas SLC4A , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética
16.
Proc Natl Acad Sci U S A ; 110(6): 2163-8, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23341620

RESUMO

Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H(+)-ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl(-) permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (Slc4a2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2's long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Calpaína/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Osteoclastos/metabolismo , Animais , Proteínas de Transporte de Ânions/deficiência , Proteínas de Transporte de Ânions/genética , Antiporters/deficiência , Antiporters/genética , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/deficiência , Antiportadores de Cloreto-Bicarbonato/genética , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Osteoclastos/patologia , Osteopetrose/genética , Osteopetrose/metabolismo , Osteopetrose/patologia , Proteínas SLC4A
17.
J Cell Biochem ; 114(3): 658-68, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23059814

RESUMO

Bone lengthening during skeletal growth is driven primarily by the controlled enlargement of growth plate (GP) chondrocytes. The cellular mechanisms are unclear but membrane transporters are probably involved. We investigated the role of the Na(+)/H(+) antiporter (NHE1) and anion exchanger (AE2) in bone lengthening and GP chondrocyte hypertrophy in Sprague-Dawley 7-day-old rat (P7) bone rudiments using the inhibitors EIPA (5-(N-ethyl-N-isopropyl)amiloride) and DIDS (4,4-diidothiocyano-2,2-stilbenedisulphonate), respectively. We have also determined cell-associated levels of these transporters along the GP using fluorescent immunohistochemistry (FIHC). Culture of bones with EIPA or DIDS inhibited rudiment growth (50% at approx. 250 and 25 µM, respectively). Both decreased the size of the hypertrophic zone (P < 0.05) but had no effect on overall length or cell density of the GP. In situ chondrocyte volume in proliferative and hypertrophic zones was decreased (P < 0.01) with EIPA but not DIDS. FIHC labeling of NHE1 was relatively high and constant along the GP but declined steeply in the late hypertrophic zone. In contrast, AE2 labeling was relatively low in proliferative zone cells but increased (P < 0.05) reaching a maximum in the early hypertrophic zone, before falling rapidly in the late hypertrophic zone suggesting AE2 might regulate the transition phase of chondrocytes between proliferative and hypertrophic zones. The inhibition of bone growth by EIPA may be due to a reduction to chondrocyte volume set-point. However the effect of DIDS was unclear but could result from inhibition of AE2 and blocking of the transition phase. These results demonstrate that NHE1 and AE2 are important regulators of bone growth.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Desenvolvimento Ósseo/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Osteogênese/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/enzimologia , Osso e Ossos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Lâmina de Crescimento/citologia , Lâmina de Crescimento/efeitos dos fármacos , Moduladores de Transporte de Membrana/antagonistas & inibidores , Moduladores de Transporte de Membrana/metabolismo , Ossos do Metatarso , Técnicas de Cultura de Órgãos , Osteogênese/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas SLC4A , Trocador 1 de Sódio-Hidrogênio
18.
Hepatology ; 57(3): 1130-41, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23080076

RESUMO

UNLABELLED: Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3',5'-monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl(-) /HCO 3- AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic bile-duct-ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down-regulation of cAMP-dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N-acetyltransferase (AANAT; the rate-limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl(-) /HCO 3- AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl(-) /HCO 3- AE2 and ablated secretin-stimulated biliary secretion in these cells. CONCLUSION: Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013).


Assuntos
Arilalquilamina N-Acetiltransferase/metabolismo , Comunicação Autócrina/fisiologia , Ductos Biliares Intra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/enzimologia , Colestase/metabolismo , Colestase/patologia , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antiporters/genética , Antiporters/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Arilalquilamina N-Acetiltransferase/genética , Comunicação Autócrina/efeitos dos fármacos , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células , Técnicas de Silenciamento de Genes , Masculino , Melatonina/sangue , Melatonina/farmacologia , Camundongos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Endogâmicos F344 , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Proteínas SLC4A
19.
FEBS J ; 280(1): 174-83, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23121767

RESUMO

The essential anion exchanger (AE) involved in bicarbonate secretion is AE2/SLC4A2, a membrane protein recognized to be relevant for the regulation of the intracellular pH in several cell types. Here we report that gastrin, a major gastrointestinal hormone, upregulates the expression of AE2 mRNA and protein in a cholecystokinin B receptor dependent manner in gastric cancer cells. The upregulated species of AE2 mRNA originates from the classical upstream promoter of the AE2 gene (here referred to as AE2a1) which provides the binding site for transcription factors early growth response 1 (EGR1) and SP1. EGR1 upregulated the AE2 expression that can be competitively inhibited by SP1 in co-transfection experiments. This competitive inhibition was avoided in cells because the SP1 expression was time-staggered to EGR1 in response to gastrin. Overexpression or knockdown of EGR1 consistently increased or decreased the expression of AE2. Our data linked a novel signal pathway involved in gastrin-stimulated AE2 expression.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Gastrinas/fisiologia , Regulação para Cima , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Antiportadores de Cloreto-Bicarbonato , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor de Colecistocinina B/metabolismo , Proteínas SLC4A , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Neoplasias Gástricas
20.
J Physiol ; 590(21): 5299-316, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22802585

RESUMO

Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl(-)/HCO(3)(-) exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (I(sc)). We have studied the role of AE2 in Cl(-) and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced 90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na(+)-K(+)-2Cl(-) cotransporter) or NBCe1 (Na(+)-nHCO(3)(-) cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO(3)(-)] as compared with the control lines. Unstimulated equivalent short-circuit current (I(eq)) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both I(eq) and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl(-)/HCO(3)(-) exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl(-) removal was reduced by ∼80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl(-) loading during cAMP-stimulated secretion of Cl(-) and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway submucosal glands and some other epithelia.


Assuntos
Proteínas de Transporte de Ânions/fisiologia , Antiporters/fisiologia , Células Epiteliais/metabolismo , Líquido Intracelular/metabolismo , Bicarbonatos/metabolismo , Bumetanida/farmacologia , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato , Cloretos/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Transporte de Íons , RNA Interferente Pequeno/genética , Sistema Respiratório/citologia , Sistema Respiratório/metabolismo , Proteínas SLC4A , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia
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