Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.040
Filtrar
1.
Int J Mol Sci ; 25(20)2024 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-39456989

RESUMO

Malabsorption of NaCl is the primary cause of diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption occurs via the dual operation of Na:H and Cl:HCO3 exchange in the brush border membrane (BBM) of villus cells. Cl:HCO3 exchange is mediated by BBM transporters DRA (downregulated in adenoma) and PAT1 (putative anion transporter 1) in the mammalian small intestine. DRA/PAT1-mediated Cl:HCO3 exchange was significantly downregulated in the BBM of villus cells in a rabbit model of chronic ileitis, while Na:H exchange was unaffected. The inhibition of Cl:HCO3 exchange was restored in the rabbits when treated with a broad-spectrum immunomodulator, i.e. a glucocorticoid, indicating that the downregulation of DRA/PAT1 is likely to be immune-mediated during chronic enteritis. Mucosal mast cells are one type of key immune cells that are known to proliferate and release immune inflammatory mediators, thus playing a significant role in the pathogenesis of IBD. However, how mast cells may regulate DRA- and PAT1-mediated Cl:HCO3 exchange in a rabbit model of chronic ileitis is unknown. In this study, treatment of rabbits with chronic intestinal inflammation with the mast cell stabilizer ketotifen did not affect the mucosal architecture of the inflamed intestine. However, ketotifen treatment reversed the inhibition of Cl:HCO3 activity in the BBM of villus cells. This restoration of Cl:HCO3 activity to normal levels by ketotifen was found to be secondary to restoring the affinity of the exchangers for its substrate chloride. This observation was consistent with molecular studies, where the mRNA and BBM protein expressions of DRA and PAT1 remained unaffected in the villus cells under all experimental conditions. Thus, this study indicates that mast cells mediated the inhibition of coupled NaCl absorption by inhibiting Cl:HCO3 exchange in a rabbit model of chronic enteritis.


Assuntos
Antiportadores de Cloreto-Bicarbonato , Ileíte , Mastócitos , Microvilosidades , Animais , Coelhos , Mastócitos/metabolismo , Microvilosidades/metabolismo , Microvilosidades/patologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Ileíte/metabolismo , Ileíte/patologia , Doença Crônica , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Bicarbonatos/metabolismo , Modelos Animais de Doenças
2.
Am J Physiol Gastrointest Liver Physiol ; 327(5): G655-G672, 2024 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-39104321

RESUMO

In this study, we uncovered the novel mechanism of IL-1α-mediated downregulated in adenoma (DRA) (SLC26A3) downregulation in the context of Brachyspira spp.-induced malabsorptive diarrhea. Experimentally infected pigs with Brachyspira spp. had significantly reduced DRA expression in the colon accompanied by IL-1α upregulation. This response was recapitulated in vitro by exposing Caco-2 cells to either Brachyspira lysate or IL-1α. Both p38 and MAPK-activated protein kinase 2 (MAPKAPK-2 also referred as MK-2) showed an increased phosphorylation after exposure to either. SB203580 application, a p38 inhibitor blocked the MK-2 phosphorylation and attenuated the DRA and IL-1α response to both lysate and IL-1α. Exposure to IL-1 receptor antagonist (IL-1RA) produced a similar response. In addition, exposure of cells to either of these blockers without IL-1α or lysate results in increased DRA and decreased IL-1α expression, revealing that DRA needs IL-1α signaling for basal physiological expression. Dual inhibition with both blockers completely inhibited the effect from IL-1α while significantly attenuating the response from Brachyspira lysate, suggesting a minor contribution from another pathway. Together this demonstrates that Brachyspira activates p38 MAPK signaling driving IL-1α expression, which activates IL-1R1 causing DRA downregulation while also driving upregulation of IL-1α through p38 in a positive feedback mechanism. In conclusion, we elucidated a major pathway involved in DRA downregulation and its role in Brachyspira-induced diarrhea. In addition, these observations will aid in our understanding of other inflammatory and infectious diarrhea conditions.NEW & NOTEWORTHY The diarrheal disease caused by the two infectious spirochete spp. B. hyodysenteriae and B. hampsonii reduced the expression of DRA (SLC26A3), a major Cl-/HCO-3 exchanger involved in Cl- absorption. This is attributed to the upregulation of IL-1α driven by p38 MAPK. This work also describes a potential new mechanism in inflammatory diseases while showing the importance of IL-1α in maintaining DRA levels.


Assuntos
Diarreia , Interleucina-1alfa , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Diarreia/microbiologia , Diarreia/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Humanos , Suínos , Células CACO-2 , Interleucina-1alfa/metabolismo , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/genética , Colo/metabolismo , Colo/microbiologia , Regulação para Baixo , Fosforilação , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Piridinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Antiportadores de Cloreto-Bicarbonato , Peptídeos e Proteínas de Sinalização Intracelular
3.
JCI Insight ; 9(14)2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38869953

RESUMO

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.


Assuntos
Bicarbonatos , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Duodeno , Camundongos Knockout , Peptídeos , Transportadores de Sulfato , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Animais , Camundongos , Bicarbonatos/metabolismo , Humanos , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/genética , Peptídeos/farmacologia , Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Duodeno/metabolismo , Duodeno/efeitos dos fármacos , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Masculino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Antiporters , Antiportadores de Cloreto-Bicarbonato
4.
BMC Pediatr ; 24(1): 305, 2024 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-38704545

RESUMO

INTRODUCTION: Congenital chloride diarrhoea (CCD) is an autosomal recessive condition that causes secretory diarrhoea and potentially deadly electrolyte imbalances in infants because of solute carrier family 26 member 3 (SLC26A3) gene mutations. CASE PRESENTATION: A 7-month-old Chinese infant with a history of maternal polyhydramnios presented with frequent watery diarrhoea, severe dehydration, hypokalaemia, hyponatraemia, failure to thrive, metabolic alkalosis, hyperreninaemia, and hyperaldosteronaemia. Genetic testing revealed a compound heterozygous SLC26A3 gene mutation in this patient (c.269_270dup and c.2006 C > A). Therapy was administered in the form of oral sodium and potassium chloride supplements, which decreased stool frequency. CONCLUSIONS: CCD should be considered when an infant presents with prolonged diarrhoea during infancy, particularly in the context of maternal polyhydramnios and dilated foetal bowel loops.


Assuntos
Diarreia , Erros Inatos do Metabolismo , Mutação , Transportadores de Sulfato , Feminino , Humanos , Lactente , Masculino , Antiportadores de Cloreto-Bicarbonato/genética , Diarreia/congênito , Diarreia/genética , População do Leste Asiático , Heterozigoto , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/diagnóstico , Poli-Hidrâmnios/genética , Cloreto de Potássio/uso terapêutico , Cloreto de Potássio/administração & dosagem , Transportadores de Sulfato/genética
5.
Int J Mol Sci ; 25(8)2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38674160

RESUMO

Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.


Assuntos
Potássio , Simportadores de Sódio-Bicarbonato , Animais , Camundongos , Bicarbonatos/metabolismo , Sítios de Ligação , Antiportadores de Cloreto-Bicarbonato/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Cloretos/metabolismo , Transporte de Íons , Simulação de Dinâmica Molecular , Potássio/metabolismo , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Sódio-Bicarbonato/genética
6.
Int J Mol Sci ; 25(8)2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38673775

RESUMO

Solute carrier family 26 member 4 (SLC26A4) is a member of the SLC26A transporter family and is expressed in various tissues, including the airway epithelium, kidney, thyroid, and tumors. It transports various ions, including bicarbonate, chloride, iodine, and oxalate. As a multiple-ion transporter, SLC26A4 is involved in the maintenance of hearing function, renal function, blood pressure, and hormone and pH regulation. In this review, we have summarized the various functions of SLC26A4 in multiple tissues and organs. Moreover, the relationships between SLC26A4 and other channels, such as cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and sodium chloride cotransporter, are highlighted. Although the modulation of SLC26A4 is critical for recovery from malfunctions of various organs, development of specific inducers or agonists of SLC26A4 remains challenging. This review contributes to providing a better understanding of the role of SLC26A4 and development of therapeutic approaches for the SLC26A4-associated hearing loss and SLC26A4-related dysfunction of various organs.


Assuntos
Transportadores de Sulfato , Humanos , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/genética , Animais , Rim/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Especificidade de Órgãos , Cloretos/metabolismo , Transporte de Íons
7.
Pflugers Arch ; 476(4): 555-564, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38195948

RESUMO

The kidney plays a crucial role in acid-base homeostasis. In the distal nephron, α-intercalated cells contribute to urinary acid (H+) secretion and ß-intercalated cells accomplish urinary base (HCO3-) secretion. ß-intercalated cells regulate the acid base status through modulation of the apical Cl-/HCO3- exchanger pendrin (SLC26A4) activity. In this review, we summarize and discuss our current knowledge of the physiological role of the renal transporter AE4 (SLC4A9). The AE4, as cation-dependent Cl-/HCO3- exchanger, is exclusively expressed in the basolateral membrane of ß-intercalated cells and is essential for the sensing of metabolic acid-base disturbances in mice, but not for renal sodium reabsorption and plasma volume control. Potential intracellular signaling pathways are discussed that might link basolateral acid-base sensing through the AE4 to apical pendrin activity.


Assuntos
Túbulos Renais Coletores , Animais , Camundongos , Antiportadores de Cloreto-Bicarbonato/metabolismo , Rim/metabolismo , Túbulos Renais Coletores/metabolismo
8.
Am J Physiol Cell Physiol ; 326(3): C829-C842, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38223928

RESUMO

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.


Assuntos
Cloretos , Diarreia , Humanos , Animais , Camundongos , Trocador 3 de Sódio-Hidrogênio/genética , Ânions , Enterócitos , Concentração de Íons de Hidrogênio , Transportadores de Sulfato/genética , Antiportadores de Cloreto-Bicarbonato/genética
9.
Theriogenology ; 217: 136-142, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38277795

RESUMO

Regulation of intracellular pH (pHi) is an important homeostatic function of cells. There are three major pHi regulatory mechanisms: the HCO3-/Cl- exchanger (AE), which alleviates alkalosis, and the Na+/H+ exchanger (NHE) and Na+,HCO3-/Cl- exchanger (NDBCE), both of which counteract acidosis. NHE activity, which is high at the germinal vesicle stage of oocyte, is inhibited during meiotic maturation, while this inhibition is abolished when the oocyte reaches the pronuclear (PN) stage of the zygote. On the other hand, we have previously found that NDBCE performs complementary regulation against acidosis during meiotic maturation. Additionally, we found that AE activity, which is a defense mechanism against alkalosis, gradually decreases during preimplantation period of embryonic development. Considering that NHE activity is inhibited during meiotic maturation and AE activity gradually decreases during embryonic development stages, we investigated whether NHE and NDBCE activities, both of which act against acidosis, functionally change from the PN zygote to the blastocyst stage of the embryo and identified these pH-regulating proteins at the molecular level in mice of the Balb/c strain. PN zygotes, two-cell (2-c), four-cell (4-c), morula and blastocyst stage embryos were obtained from 5-8-week-old, sexually mature female Balb/c mice by using the classical superovulation procedure. pHi was recorded by using the microspectrofluorometric technique on zygotes and embryos simultaneously loaded with the pH-sensitive fluorophore, 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The activities of NHE and NDBCE were determined from the recovery curve of induced-acidosis in bicarbonate-free and bicarbonate-containing media, respectively. Specific inhibitors such as cariporide (1 µM), S3226 (1 and 10 µM), EIPA (1, 5, and 25 µM), and amiloride (1 mM) were used to functionally identify NHE isoforms, and the nonspecific inhibitor 4,4'-diisocyanatostilbene-2,2' disulphonic acid, disodium salt (DIDS) was used to confirm NDBCE activity. The isoforms of the pHi-regulatory proteins were also identified by molecular biology using real-time PCR. We found that NHE activity was high at all embryonic stages, and differences between stages were not significant. Functional and molecular findings indicated that isoforms of NHE 1 and 5 are present in the blastocyst, whereas isoforms of NHE 1, 3, and 4 are functional at earlier embryonic stages. Although the contribution of NDBCE activity to recovery from induced-acidosis was detected at all embryonic stages, it was significant only in the PN zygote and the 2-c embryo. This finding was confirmed by molecular analysis, which detected the expression of SLC4A8 encoding NDBCE at all embryonic stages. In conclusion, NHE is an active and important defense mechanism against acidosis and is encoded by at least two protein isoforms in all stages of the Balb/c strain of mice. NDBCE has a supportive function in all embryonic stages, especially in the PN zygote and the 2-c embryo. Preimplantation stage embryos have effective mechanisms to defend against acidosis in response to their metabolic end products (increased acid load) and the acidic environment in utero.


Assuntos
Acidose , Alcalose , Doenças dos Roedores , Gravidez , Camundongos , Feminino , Animais , Concentração de Íons de Hidrogênio , Antiportadores de Cloreto-Bicarbonato/fisiologia , Camundongos Endogâmicos BALB C , Acidose/veterinária , Trocadores de Sódio-Hidrogênio/metabolismo , Alcalose/veterinária , Isoformas de Proteínas/metabolismo , Mecanismos de Defesa
10.
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
11.
Genet Med ; 26(3): 101034, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38054405

RESUMO

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.


Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Animais , Humanos , Camundongos , Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Deficiência Intelectual/genética , Proteínas de Membrana Transportadoras , Camundongos Knockout , Transtornos do Neurodesenvolvimento/genética , Sódio/metabolismo , Bicarbonato de Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/genética
12.
Brain ; 146(11): 4547-4561, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37459438

RESUMO

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.


Assuntos
Convulsões , Simportadores de Sódio-Bicarbonato , Criança , Camundongos , Humanos , Animais , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Convulsões/genética , Mutação/genética , Neurotransmissores , Ácido gama-Aminobutírico/genética , Mamíferos/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo
13.
Cell Rep ; 42(6): 112601, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37270778

RESUMO

Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.


Assuntos
Antiportadores de Cloreto-Bicarbonato , Proteínas de Membrana Transportadoras , Neoplasias , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato/química , Antiportadores de Cloreto-Bicarbonato/metabolismo , Citoplasma/metabolismo , Concentração de Íons de Hidrogênio , Neoplasias/metabolismo , Humanos
14.
Elife ; 122023 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-37351578

RESUMO

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the 10 functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter et al., 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.


Assuntos
Antiporters , Bicarbonatos , Humanos , Animais , Camundongos , Antiporters/metabolismo , Bicarbonatos/metabolismo , Cloretos/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Transportadores de Sulfato/genética
15.
Int J Neuropsychopharmacol ; 26(6): 396-411, 2023 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-37235790

RESUMO

BACKGROUND: Major depressive disorder (MDD) is a type of emotional dysfunction, and its pathogenesis has not been fully elucidated. Specifically, the key molecules in depression-related brain regions involved in this disease and their contributions to this disease are currently unclear. METHODS: GSE53987 and GSE54568 were selected from the Gene Expression Omnibus database. The data were standardized to identify the common differentially expressed genes (DEGs) in the cortex of MDD patients in the 2 datasets. The DEGs were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. The STRING database was used to build protein-protein interaction networks, and the cytoHubba plugin was used to identify hub genes. Furthermore, we selected another blood transcriptome dataset that included 161 MDD and 169 control samples to explore the changes in the screened hub genes. Mice were subjected to 4 weeks of chronic unpredictable mild stress to establish an animal model of depression, and the expression of these hub genes in tissues of the prefrontal cortex was then detected by quantitative real time polymerase chain reaction (qRT-PCR). We subsequently predicted the possible posttranscriptional regulatory networks and traditional Chinese medicine according to the hub genes using a few online databases. RESULTS: The analysis identified 147 upregulated genes and 402 downregulated genes were identified in the cortex of MDD patients compared with that of the controls. Enrichment analyses revealed that DEGs were predominantly enriched in synapse-related cell functions, linoleic acid metabolism, and other pathways. Protein-protein interaction analysis identified 20 hub genes based on the total score. The changes in KDM6B, CUX2, NAAA, PHKB, NFYA, GTF2H1, CRK, CCNG2, ACER3, and SLC4A2 in the peripheral blood of MDD patients were consistent with those in the brain. Furthermore, the prefrontal cortex of mice with depressive-like behaviors showed significantly increased Kdm6b, Aridb1, Scaf11, and Thoc2 expression and decreased Ccng2 expression compared with that of normal mice, which was consistent with the results found for the human brain. Potential therapeutic candidates, such as citron, fructus citri, leaves of Panax Notoginseng, sanchi flower, pseudoginseng, and dan-shen root, were selected via traditional Chinese medicine screening. CONCLUSIONS: This study identified several novel hub genes in specific brain regions involved in the pathogenesis of MDD, which may not only deepen our understanding of depression but may also provide new ideas for its diagnosis and treatment.


Assuntos
Transtorno Depressivo Maior , Humanos , Animais , Camundongos , Transtorno Depressivo Maior/genética , Redes Reguladoras de Genes , Perfilação da Expressão Gênica/métodos , Mapas de Interação de Proteínas , Encéfalo , Biologia Computacional/métodos , Fator de Transcrição TFIIH/genética , Histona Desmetilases com o Domínio Jumonji/genética , Antiportadores de Cloreto-Bicarbonato/genética
16.
Nat Commun ; 14(1): 3051, 2023 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-37236964

RESUMO

The kidney plays a key role in the correction of systemic acid-base imbalances. Central for this regulation are the intercalated cells in the distal nephron, which secrete acid or base into the urine. How these cells sense acid-base disturbances is a long-standing question. Intercalated cells exclusively express the Na+-dependent Cl-/HCO3- exchanger AE4 (Slc4a9). Here we show that AE4-deficient mice exhibit a major dysregulation of acid-base balance. By combining molecular, imaging, biochemical and integrative approaches, we demonstrate that AE4-deficient mice are unable to sense and appropriately correct metabolic alkalosis and acidosis. Mechanistically, a lack of adaptive base secretion via the Cl-/HCO3- exchanger pendrin (Slc26a4) is the key cellular cause of this derailment. Our findings identify AE4 as an essential part of the renal sensing mechanism for changes in acid-base status.


Assuntos
Rim , Proteínas de Membrana Transportadoras , Camundongos , Animais , Rim/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Equilíbrio Ácido-Base/fisiologia , Néfrons/metabolismo , Transportadores de Sulfato/metabolismo , Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato
17.
Am J Physiol Cell Physiol ; 324(6): C1263-C1273, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37154494

RESUMO

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl-/[Formula: see text] exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+i) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca2+i, followed by 0.25 µM ATP produced a large increase in Ca2+ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca2+i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases, such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, whereas separation of DRA from Na+/H+ exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption.NEW & NOTEWORTHY The BB Cl-/[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca2+. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca2+ are involved.


Assuntos
Células Epiteliais , Cloreto de Sódio , Humanos , Células CACO-2 , Células Epiteliais/metabolismo , Ânions/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Diarreia/metabolismo , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo
18.
Int J Mol Sci ; 24(9)2023 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-37175979

RESUMO

Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl-/HCO3- antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air-liquid interface (ALI) was created by removing apical media for a specified number of days; from 12-14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids.


Assuntos
Antiporters , Lipídeos de Membrana , Humanos , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/metabolismo , Antiporters/metabolismo , Diferenciação Celular , Transportadores de Sulfato/metabolismo
19.
Genes (Basel) ; 14(3)2023 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-36980855

RESUMO

The Luxi gamecock developed very unique morphological and behavioral features under the special artificial selection of the most famous Chinese gamecocks. There are very few research studies on the genetics and selection of the Luxi gamecock. We used six methods (Fst, Tajima's D, hapFLK, iHS, XP-EHH, and Runs of homozygosity) to detect selective sweeps in whole-genome resequencing data of 19 Luxi gamecocks compared to other Chinese indigenous chickens. Eleven genes that were highly related to nervous system development (CDH18, SLITRK1, SLITRK6, NDST3, ATP23, LRIG3, IL1RAPL1, GADL1, C5orf22, UGT8, WISP1, and WNT9A) appeared in at least four methods and were regarded as the most significant genes under selection. Differentially expressed gene (DEG) analysis based on the RNA sequencing data of the cerebral cortex and middle brain between six Luxi gamecocks, Tibetan chickens, and white leghorns found that most differentially expressed genes were enriched in pathways with nervous system functions. Genes associated with aggressiveness-related neurotransmitters (SLC4A2, DRD1, DRD2, ADRA2A, and ADRA2B) showed differential expression rates in Luxi gamecocks as well. Combined results showed that most genes in selective sweep regions were also differentially expressed in Luxi gamecocks including the most significant genes (SLITRK6, IL1RAPL1, GADL1, WISP1, and LRIG3). This study provides more insight into molecular mechanisms of the aggressiveness of gamecocks and aims to promote further studies on animal and human aggression.


Assuntos
Carboxiliases , Galinhas , Animais , Humanos , Galinhas/genética , Sequência de Bases , Análise de Sequência de DNA , Análise de Sequência de RNA , Sistema Nervoso , Proteínas de Membrana/genética , Antiportadores de Cloreto-Bicarbonato/genética , Carboxiliases/genética
20.
Nat Commun ; 14(1): 1812, 2023 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-37002221

RESUMO

The cell maintains its intracellular pH in a narrow physiological range and disrupting the pH-homeostasis could cause dysfunctional metabolic states. Anion exchanger 2 (AE2) works at high cellular pH to catalyze the exchange between the intracellular HCO3- and extracellular Cl-, thereby maintaining the pH-homeostasis. Here, we determine the cryo-EM structures of human AE2 in five major operating states and one transitional hybrid state. Among those states, the AE2 shows the inward-facing, outward-facing, and intermediate conformations, as well as the substrate-binding pockets at two sides of the cell membrane. Furthermore, critical structural features were identified showing an interlock mechanism for interactions among the cytoplasmic N-terminal domain and the transmembrane domain and the self-inhibitory effect of the C-terminal loop. The structural and cell-based functional assay collectively demonstrate the dynamic process of the anion exchange across membranes and provide the structural basis for the pH-sensitive pH-rebalancing activity of AE2.


Assuntos
Proteínas de Transporte de Ânions , Antiporters , Humanos , Antiportadores de Cloreto-Bicarbonato , Concentração de Íons de Hidrogênio , Membrana Celular/metabolismo , Homeostase , Antiporters/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Cloretos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA