RESUMO
Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.
Assuntos
Cavéolas , Traumatismo por Reperfusão , Ratos , Animais , Cavéolas/metabolismo , Actinas/metabolismo , Caveolina 3/metabolismo , Citoesqueleto/metabolismo , Caveolina 1/metabolismo , Traumatismo por Reperfusão/metabolismo , Mitocôndrias/metabolismoRESUMO
The role of Cl- as an intracellular signaling ion has been increasingly recognized in recent years. One of the currently best described roles of Cl- in signaling is the modulation of the With-No-Lysine (K) (WNK) - STE20-Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1) - Cation-Coupled Cl- Cotransporters (CCCs) cascade. Binding of a Cl- anion to the active site of WNK kinases directly modulates their activity, promoting their inhibition. WNK activation due to Cl- release from the binding site leads to phosphorylation and activation of SPAK/OSR1, which in turn phosphorylate the CCCs. Phosphorylation by WNKs-SPAK/OSR1 of the Na+-driven CCCs (mediating ions influx) promote their activation, whereas that of the K+-driven CCCs (mediating ions efflux) promote their inhibition. This results in net Cl- influx and feedback inhibition of WNK kinases. A wide variety of alterations to this pathway have been recognized as the cause of several human diseases, with manifestations in different systems. The understanding of WNK kinases as Cl- sensitive proteins has allowed us to better understand the mechanistic details of regulatory processes involved in diverse physiological phenomena that are reviewed here. These include cell volume regulation, potassium sensing and intracellular signaling in the renal distal convoluted tubule, and regulation of the neuronal response to the neurotransmitter GABA.
RESUMO
The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. The functional and structural characterization of NCC from different species has led us to gain insights into the structure-function relationships of the cotransporter. Here we present an overview of different studies that had described these properties. Additionally, we report the cloning and characterization of the NCC from the spiny dogfish (Squalus acanthias) kidney (sNCC). The purpose of the present study was to determine the main functional, pharmacological and regulatory properties of sNCC to make a direct comparison with other NCC orthologous. The sNCC cRNA encodes a 1033 amino acid membrane protein, when expressed in Xenopus oocytes, functions as a thiazide-sensitive Na-Cl cotransporter with NCC regulation and thiazide-inhibition properties similar to mammals, rather than to teleosts. However, the Km values for ion transport kinetics are significantly higher than those observed in the mammal species. In summary, we present a review on NCC structure-function relationships with the addition of the sNCC information in order to enrich the NCC cotransporter knowledge.
Assuntos
Rim/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/química , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Síndrome de Gitelman/genética , Humanos , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Relação Estrutura-AtividadeRESUMO
Ion Transport across the cell membrane is required to maintain cell volume homeostasis. In response to changes in extracellular osmolarity, most cells activate specific metabolic or membrane-transport pathways to respond to cell swelling or shrinkage and return their volume to its normal resting state. This process involves the rapid adjustment of the activities of channels and transporters that mediate flux of K+, Na+, Cl-, and small organic osmolytes. Cation chloride cotransporters (CCCs) NKCCs and KCCs are a family of membrane proteins modulated by changes in cell volume and/or in the intracellular chloride concentration ([Cl-]i). Cell swelling triggers regulatory volume decrease (RVD), promoting solute and water efflux to restore normal cell volume. Swelling-activated KCCs mediate RVD in most cell types. In contrast, cell shrinkage triggers regulatory volume increase (RVI), which involves the activation of the NKCC1 cotransporter of the CCC family. Regulation of the CCCs during RVI and RVD by protein phosphorylation is a well-characterized mechanism, where WNK kinases and their downstream kinase substrates, SPAK and OSR1 constitute the essential phospho-regulators. WNKs-SPAK/OSR1-CCCs complex is required to regulate cell shrinkage-induced RVI or cell swelling-induced RVD via activating or inhibitory phosphorylation of NKCCs or KCCs, respectively. WNK1 and WNK4 kinases have been established as [Cl-]i sensors/regulators, while a role for WNK3 kinase as a cell volume-sensing kinase has emerged and is proposed in this chapter.
Assuntos
Tamanho Celular , Animais , Cloretos/metabolismo , Humanos , Transporte de Íons/fisiologia , Fosforilação , Sódio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismoRESUMO
Endomorphins (EMs) have been proposed as the endogenous ligand agonists of the µ-opioid receptor; however, no propeptide precursor protein for EMs has been identified. Here, to identify the presumed precursor of EMs, we designed an immunoscreening assay using specific affinity-purified rabbit antisera raised against synthetic EMs in a whole-mouse brain cDNA library. Following this approach, we identify a DNA sequence encoding a protein precursor, which we name proMexneurin, that contains three different peptide sequences: Mexneurin-1 (an EM-like peptide), Mexneurin-2, and Mexneurin-3, a peptide which appears to be unrelated to EMs. RT-PCR analysis and in situ hybridization reveal a widespread distribution of proMexneurin mRNA throughout the mouse brain. Both Mexneurin-1 and Mexneurin-3 peptides display biological activities in the mouse CNS.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/citologia , Encéfalo/fisiologia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Potenciais Evocados , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Ligantes , Masculino , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/fisiologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fases de Leitura Aberta , Técnicas de Patch-Clamp , Precursores de Proteínas/química , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteólise , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Análise de Sequência de DNARESUMO
Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation - a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl--sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl- uptake and stimulation of KCC3-mediated Cl- extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought "Cl-/volume-sensitive kinase" of the cation-Cl- cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.
Assuntos
Encéfalo/metabolismo , Cloretos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores/metabolismo , Animais , Barreira Hematoencefálica , Tamanho Celular , Células HEK293 , Humanos , Transporte de Íons , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pressão Osmótica , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteômica , Interferência de RNA , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/deficiência , Simportadores/genéticaRESUMO
The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Animais , Tamanho Celular , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina , Antígenos de Histocompatibilidade Menor/genética , Mutação , Osmorregulação , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Transfecção , Proteína Quinase 1 Deficiente de Lisina WNK , Xenopus laevis , Cotransportadores de K e Cl-RESUMO
Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.
Assuntos
Estradiol/metabolismo , Rim/metabolismo , Ovário/metabolismo , Progesterona/metabolismo , Prolactina/metabolismo , Animais , Estradiol/administração & dosagem , Terapia de Reposição de Estrogênios , Feminino , Humanos , Isoenzimas , Rim/efeitos dos fármacos , Masculino , Camundongos Knockout , Ovariectomia , Fosforilação , Progesterona/administração & dosagem , Prolactina/administração & dosagem , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Wistar , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Fatores Sexuais , Transdução de Sinais , Membro 3 da Família 12 de Carreador de Soluto/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Regulação para CimaRESUMO
Precise homoeostasis of the intracellular concentration of Cl- is achieved via the co-ordinated activities of the Cl- influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+-K+ ion co-transporters), also promote inhibition of the KCCs (K+-Cl- co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl- influx, we propose that the targeting of WNK-SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl- extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.
Assuntos
Cloretos/metabolismo , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Dados de Sequência Molecular , Fosfopeptídeos/metabolismo , Fosforilação , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Simportadores de Cloreto de Sódio-Potássio/metabolismoRESUMO
The K(+):Cl(-) cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.
Assuntos
Pressão Osmótica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Oócitos/citologia , Oócitos/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Serina/genética , Serina/metabolismo , Simportadores/genética , Xenopus laevisRESUMO
The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Sítios de Ligação , Células HEK293 , Humanos , Mutação , Oócitos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
Mouse protein-25 (MO25) isoforms bind to the STRAD pseudokinase and stabilise it in a conformation that can activate the LKB1 tumour suppressor kinase. We demonstrate that by binding to several STE20 family kinases, MO25 has roles beyond controlling LKB1. These new MO25 targets are SPAK/OSR1 kinases, regulators of ion homeostasis and blood pressure, and MST3/MST4/YSK1, involved in controlling development and morphogenesis. Our analyses suggest that MO25α and MO25ß associate with these STE20 kinases in a similar manner to STRAD. MO25 isoforms induce approximately 100-fold activation of SPAK/OSR1 dramatically enhancing their ability to phosphorylate the ion cotransporters NKCC1, NKCC2 and NCC, leading to the identification of several new phosphorylation sites. siRNA-mediated reduction of expression of MO25 isoforms in mammalian cells inhibited phosphorylation of endogenous NKCC1 at residues phosphorylated by SPAK/OSR1, which is rescued by re-expression of MO25α. MO25α/ß binding to MST3/MST4/YSK1 also stimulated kinase activity three- to four-fold. MO25 has evolved as a key regulator of a group of STE20 kinases and may represent an ancestral mechanism of regulating conformation of pseudokinases and activating catalytically competent protein kinases.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Homeostase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Morfogênese/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Ativação Enzimática/fisiologia , Escherichia coli , Células HEK293 , Humanos , Immunoblotting , Insetos , Fosforilação , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Ion cotransporters, such as the Na(+)/Cl(-) cotransporter (NCC), control renal salt re-absorption and are regulated by the WNK-signalling pathway, which is over-stimulated in patients suffering from Gordon's hypertension syndrome. Here, we study the regulation of the NKCC2 (SLC12A1) ion cotransporter that contributes towards ~25% of renal salt re-absorption and is inhibited by loop-diuretic hypertensive drugs. We demonstrate that hypotonic low-chloride conditions that activate the WNK1-SPAK and OSR1 pathway promote phosphorylation of NKCC2 isoforms (A, B and F) at five residues (Ser91, Thr95, Thr100, Thr105 and Ser130). We establish that the SPAK and OSR1 kinases activated by WNK interact with an RFQV motif on NKCC2 and directly phosphorylate Thr95, Thr100, Thr105 and, possibly, Ser91. Our data indicate that a SPAK-OSR1-independent kinase, perhaps AMP-activated protein kinase (AMPK), phosphorylates Ser130 and that phosphorylation of Thr105 and Ser130 plays the most important roles in stimulating NKCC2 activity. In contrast with NCC, whose membrane translocation is triggered by SPAK-OSR1 phosphorylation, NKCC2 appears to be constitutively at the membrane. Our findings provide new insights into how NKCC2 is regulated and suggest that inhibitors of SPAK and/or OSR1 for the treatment of hypertension would be therapeutically distinct from thiazide or loop diuretics, as they would suppress the activity of both NCC and NKCC2.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Células HEK293 , Humanos , Íons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Alinhamento de Sequência , Serina/metabolismo , Transdução de Sinais/fisiologia , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 1 da Família 12 de Carreador de Soluto , Treonina/metabolismoRESUMO
Two members of a recently discovered family of protein kinases are the cause of an inherited disease known as pseudohypoaldosteronism type II (PHAII). These patients exhibit arterial hypertension together with hyperkalemia and metabolic acidosis. This is a mirror image of Gitelman disease that is due to inactivating mutations of the SLC12A3 gene that encodes the thiazide-sensitive Na(+):Cl(-) cotransporter. The uncovered genes causing PHAII encode for serine/threonine kinases known as WNK1 and WNK4. Physiological and biochemical studies have revealed that WNK1 and WNK4 modulate the activity of several transport pathways of the aldosterone-sensitive distal nephron, thus increasing our understanding of how diverse renal ion transport proteins are coordinated to regulate normal blood pressure levels. Observations discussed in the present work place WNK1 and WNK4 as genes involved in the genesis of essential hypertension and as potential targets for the development of antihypertensive drugs.
Assuntos
Hipertensão/metabolismo , Rim/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Humanos , Hipertensão/genética , Peptídeos e Proteínas de Sinalização Intracelular , Transporte de Íons , Antígenos de Histocompatibilidade Menor , Néfrons/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
The Na(+):K(+):2Cl(-) cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl(-)](i). The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.
Assuntos
Cloretos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Humanos , Camundongos , Mutação , Oócitos , Fosforilação , Ratos , Simportadores de Cloreto de Sódio-Potássio/química , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 1 da Família 12 de Carreador de Soluto , Treonina/química , Treonina/genética , Treonina/metabolismo , XenopusRESUMO
SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular Cl- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl- influx and efflux branches of the SLC12A cotransporter family.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Simportadores/metabolismo , Animais , Ativação Enzimática , Feminino , Humanos , Soluções Hipotônicas , Cinética , Camundongos , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Simportadores/genética , Xenopus laevis , Cotransportadores de K e Cl-RESUMO
The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: Cl(-) influx is mediated by the Na-K-2Cl cotransporter NKCC1 and Cl(-) efflux via K-Cl cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.
Assuntos
Tamanho Celular , Cloretos/metabolismo , Transporte de Íons/fisiologia , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Hibridização In Situ , Camundongos , FosforilaçãoRESUMO
WNK1 and WNK4 [WNK, with no lysine (K)] are serine-threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased K(+) secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin.
Assuntos
Pressão Sanguínea/fisiologia , Homeostase , Rim/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Simportadores de Cloreto de Sódio-Potássio/fisiologia , Simportadores/fisiologia , Animais , Células COS , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Camundongos , Microscopia de Fluorescência , Fosforilação , Membro 1 da Família 12 de Carreador de Soluto , TransfecçãoRESUMO
The mammalian kidney bumetanide-sensitive Na(+)-K(+)-2Cl(-) and thiazide-sensitive Na(+)-Cl(-) cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters to design and study chimeric proteins in which the NH(2)-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na(+)-K(+)-2Cl(-) cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH(2)-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na(+)-Cl(-) cotransporter.