RESUMO
With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either â¼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Túbulos de Malpighi/enzimologia , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular , Cloretos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Concentração de Íons de Hidrogênio , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica , Especificidade por SubstratoRESUMO
Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule.Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.ResultsIn vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux.Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cloretos/metabolismo , Proteínas de Drosophila/metabolismo , Túbulos de Malpighi/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ligação ao Cálcio/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Epitélio/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Transporte de Íons/genética , Fosforilação , Transdução de SinaisRESUMO
In vivo recycling of nitrate (NO(3)(-)) and nitrite (NO(2)(-)) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate-nitrite-NO balance. More than 25% of the circulating NO(3)(-) is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO(3)(-) to NO(2)(-), which enters circulation and leads to NO generation. The transporters for NO(3)(-) in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO(3)(-)/H(+) cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO(3)(-) or sialic acid (SA), but not by Br(-), and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO(3)(-)-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H(+)-dependent NO(3)(-) conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO(3)(-) secretion in saliva after intake of a NO(3)(-)-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO(3)(-)/H(+) transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Membrana Celular/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Ácidos/metabolismo , Adenoviridae/metabolismo , Animais , Ânions , Transporte Biológico , Fibroblastos/metabolismo , Fibroblastos/patologia , Espaço Intracelular/metabolismo , Mutação/genética , Ácido N-Acetilneuramínico/metabolismo , Transportadores de Nitrato , Nitratos/metabolismo , Transportadores de Ânions Orgânicos/genética , Prótons , Doença do Armazenamento de Ácido Siálico/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Sus scrofa , Simportadores/genéticaRESUMO
Tropomyosin (TM) is the main allergen in shrimp (Litopenaeus vannamei). In this study, the effects of allergenicity and structure of TM by glycosylation (GOS-TM), phosphate treatment (SP-TM), and glycosylation combined with phosphate treatment (GOS-SP-TM) were investigated. Compared to GOS-TM and SP-TM, the IgG/IgE binding capacity of GOS-SP-TM was significantly decreased with 63.9 ± 2.0 and 49.7 ± 2.7%, respectively. Meanwhile, the α-helix content reduced, surface hydrophobicity increased, and 10 specific amino acids (K30, K38, S39, K48, K66, K74, K128, K161, S210, and K251) were modified by glycosylation on six IgE linear epitopes of GOS-SP-TM. In the BALB/c mice allergy model, GOS-SP-TM could significantly reduce the levels of specific IgE, IgG1, and CD4+IL-4+, while the levels of IgG2a, CD4+CD25+Foxp3+, and CD4+IFN-γ+ were increased, which equilibrated Th1 and Th2 cells, thus alleviating allergic symptoms. These results indicated that glycosylation combined with phosphate treatment can provide a new insight into developing hypoallergenic shrimp food.
Assuntos
Alérgenos , Imunoglobulina E , Penaeidae , Fosfatos , Tropomiosina , Animais , Feminino , Humanos , Camundongos , Alérgenos/imunologia , Alérgenos/química , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/química , Hipersensibilidade Alimentar/imunologia , Glicosilação , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/química , Camundongos Endogâmicos BALB C , Penaeidae/imunologia , Penaeidae/química , Fosfatos/química , Frutos do Mar/análise , Hipersensibilidade a Frutos do Mar/imunologia , Células Th2/imunologia , Células Th2/efeitos dos fármacos , Tropomiosina/imunologia , Tropomiosina/químicaRESUMO
Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies.1,2 Circadian variation in the intracellular chloride concentration of mammalian pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels.3 However, results have been contradictory,4-9 and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response.10 Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLNv) pacemaker neurons. In control flies, intracellular chloride increases in sLNvs over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations.11Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLNv pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Cloretos , Ritmo Circadiano , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mamíferos , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologiaRESUMO
The electroneutral cation-chloride cotransporter gene family, SLC12, contains nine members in vertebrates. These include seven sodium and/or potassium-coupled chloride transporters and two membrane proteins of unknown function. Although SLC12 family members have been identified in a number of lower species, the functional properties of these proteins are unknown. There are five SLC12 homologues in Drosophila melanogaster, including at least one member on each of the four main branches of the vertebrate phylogenetic tree. We have employed in situ hybridization to study the expression patterns of the Drosophila SLC12 proteins during embryonic development. Our studies indicate that all five members of this family are expressed during early embryogenesis (stages 1-6), but that spatial and temporal expression patterns become more refined as development proceeds. Expression during late embryogenesis was seen predominantly in the ventral nerve cord, salivary gland, gut, and anal pad. In parallel studies, we have carried out transport assays on each of the five Drosophila homologues, expressed as recombinant proteins in the cultured insect cell line High Five. Under our experimental conditions, we found that only one of these proteins, CG4357, transported the potassium congener (86)Rb. Additional experiments established that rubidium transport via CG4357 was saturable (K(m) = 0.29 +/- 0.05 mM), sodium-dependent (half-saturation constant = 53 +/- 11 mM), chloride-dependent (half-saturation constant = 48 +/- 5 mM), and potently inhibited by bumetanide (inhibitor constant = 1.17 +/- 0.08 muM), a specific inhibitor of Na(+)-K(+)-2Cl(-) cotransporters. Taken together, our results provide strong evidence that CG4357 is an insect ortholog of the vertebrate Na(+)-K(+)-2Cl(-) cotransporters.
Assuntos
Quimiocina CCL21/genética , Drosophila melanogaster/genética , Simportadores de Cloreto de Sódio-Potássio/genética , Canal Anal/fisiologia , Animais , Clonagem Molecular , Códon de Terminação/genética , Primers do DNA , DNA Complementar/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Família Multigênica , Fenômenos Fisiológicos do Sistema Nervoso , Sondas RNA , Mapeamento por Restrição , Rubídio/metabolismo , Rubídio/farmacologia , Glândulas Salivares/fisiologiaRESUMO
AIM: To prepare the rabbit antibody against human sialin and identify its properties. METHODS: Recombinant expression vector pGEX-5X-1-sialin was constructed, in which the sialin cDNA encoding the 1-38 aa was fused to the C-terminal of the gene encoding the GST protein. The GST-sialin (N1-38) fusion protein was expressed in E. coli JM109 at 37 degrees C in the presence of IPTG at 0.1 mmol/L for induction for 3 hours, purified by GSTrap FF, and then used as the immunogen to prepare the rabbit polyclonal antibody. The properties of antiserum against human sialin were identified by ELISA, Western blot and immunocytochemistry. RESULTS: The recombinant expression plasmid pGEX-5X-1-sialin was constructed. The GST-sialin (N1-38) fusion protein was highly expressed with a molecular weight of 30 kDa, and the yield of the fusion protein was about 20% to 30% in total E. coli protein. The titre of antiserum against human sialin was 1:32,000. Western blot analysis proved the rabbit polyclonal antibody could identify both GST-sialin (N1-38) fusion protein and GST. Besides, it specially recognized a 55 kDa band expressed in the human submandibular gland (HSG) cell line. The antigen recognized by the antibody was located in the cytoplasm and nucleus of HSG cell. CONCLUSION: The successful preparation of the polyclonal antibody against human sialin will provide efficient affinity reagent for further functional study of sialin expressed in human salivary glands.