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Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.
Koumangoye R, Omer S, Delpire E
(2018) Am J Physiol Cell Physiol 315: C258-C276
MeSH Terms: Animals, Cell Membrane, Cells, Cultured, Colon, Cytoplasm, Dogs, Epithelial Cells, Female, Madin Darby Canine Kidney Cells, Male, Mice, Oocytes, Salivary Glands, Sodium-Potassium-Chloride Symporters, Sodium-Potassium-Exchanging ATPase, Solute Carrier Family 12, Member 2, Xenopus laevis
Show Abstract · Added May 4, 2018
We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na-K-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.
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17 MeSH Terms
Na -K -2Cl Cotransporter (NKCC) Physiological Function in Nonpolarized Cells and Transporting Epithelia.
Delpire E, Gagnon KB
(2018) Compr Physiol 8: 871-901
MeSH Terms: Animals, Biological Transport, Cell Membrane, Epithelial Cells, Gene Expression Regulation, Humans, Sodium-Potassium-Chloride Symporters, Structure-Activity Relationship
Show Abstract · Added April 2, 2019
Two genes encode the Na -K -2Cl cotransporters, NKCC1 and NKCC2, that mediate the tightly coupled movement of 1Na , 1K , and 2Cl across the plasma membrane of cells. Na -K -2Cl cotransport is driven by the chemical gradient of the three ionic species across the membrane, two of them maintained by the action of the Na /K pump. In many cells, NKCC1 accumulates Cl above its electrochemical potential equilibrium, thereby facilitating Cl channel-mediated membrane depolarization. In smooth muscle cells, this depolarization facilitates the opening of voltage-sensitive Ca channels, leading to Ca influx, and cell contraction. In immature neurons, the depolarization due to a GABA-mediated Cl conductance produces an excitatory rather than inhibitory response. In many cell types that have lost water, NKCC is activated to help the cells recover their volume. This is specially the case if the cells have also lost Cl . In combination with the Na /K pump, the NKCC's move ions across various specialized epithelia. NKCC1 is involved in Cl -driven fluid secretion in many exocrine glands, such as sweat, lacrimal, salivary, stomach, pancreas, and intestine. NKCC1 is also involved in K -driven fluid secretion in inner ear, and possibly in Na -driven fluid secretion in choroid plexus. In the thick ascending limb of Henle, NKCC2 activity in combination with the Na /K pump participates in reabsorbing 30% of the glomerular-filtered Na . Overall, many critical physiological functions are maintained by the activity of the two Na -K -2Cl cotransporters. In this overview article, we focus on the functional roles of the cotransporters in nonpolarized cells and in epithelia. © 2018 American Physiological Society. Compr Physiol 8:871-901, 2018.
Copyright © 2018 American Physiological Society. All rights reserved.
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8 MeSH Terms
A patient with multisystem dysfunction carries a truncation mutation in human , the gene encoding the Na-K-2Cl cotransporter, NKCC1.
Delpire E, Wolfe L, Flores B, Koumangoye R, Schornak CC, Omer S, Pusey B, Lau C, Markello T, Adams DR
(2016) Cold Spring Harb Mol Case Stud 2: a001289
MeSH Terms: Adolescent, Alleles, Carrier Proteins, Cell Membrane, Chlorides, Female, Fibroblasts, Humans, Mutation, Sequence Deletion, Sodium, Sodium, Dietary, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 2, Whole Exome Sequencing
Show Abstract · Added April 26, 2017
This study describes a 13-yr-old girl with orthostatic intolerance, respiratory weakness, multiple endocrine abnormalities, pancreatic insufficiency, and multiorgan failure involving the gut and bladder. Exome sequencing revealed a de novo, loss-of-function allele in , the gene encoding the Na-K-2Cl cotransporter-1. The 11-bp deletion in exon 22 results in frameshift (p.Val1026Phe*2) and truncation of the carboxy-terminal tail of the cotransporter. Preliminary studies in heterologous expression systems demonstrate that the mutation leads to a nonfunctional transporter, which is expressed and trafficked to the plasma membrane alongside wild-type NKCC1. The truncated protein, visible at higher molecular sizes, indicates either enhanced dimerization or misfolded aggregate. No significant dominant-negative effect was observed. K transport experiments performed in fibroblasts from the patient showed reduced total and NKCC1-mediated K influx. The absence of a bumetanide effect on K influx in patient fibroblasts only under hypertonic conditions suggests a deficit in NKCC1 regulation. We propose that disruption in NKCC1 function might affect sensory afferents and/or smooth muscle cells, as their functions depend on NKCC1 creating a Cl gradient across the plasma membrane. This Cl gradient allows the γ-aminobutyric acid (GABA) receptor or other Cl channels to depolarize the membrane affecting processes such as neurotransmission or cell contraction. Under this hypothesis, disrupted sensory and smooth muscle function in a diverse set of tissues could explain the patient's phenotype.
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15 MeSH Terms
The KCC2 Cotransporter and Human Epilepsy: Getting Excited About Inhibition.
Kahle KT, Khanna AR, Duan J, Staley KJ, Delpire E, Poduri A
(2016) Neuroscientist 22: 555-562
MeSH Terms: Animals, Anticonvulsants, Epilepsy, Genetic Predisposition to Disease, Humans, Mutation, Sodium-Potassium-Chloride Symporters, Symporters
Show Abstract · Added May 3, 2017
The cation-Cl cotransporter KCC2, encoded by SLC12A5, is required for the emergence and maintenance of GABAergic fast synaptic inhibition in organisms across evolution. These findings have suggested that KCC2 deficiency might play a role in the pathogenesis human epilepsy, but this has only recently been substantiated by two lines of genetic evidence. The first is the discovery of heterozygous missense polymorphisms in SLC12A5, causing decreased KCC2-dependent Cl extrusion capacity, in an Australian family with inherited febrile seizures and in a French-Canadian cohort with severe genetic generalized epilepsy (GGE). The second is the discovery of recessive loss-of-function mutations in SLC12A5 in patients with a severe, early-onset Mendelian disease termed "epilepsy of infancy with migrating focal seizures" (EIMFS). These findings collectively support the paradigm that precisely regulated KCC2 activity is required for synaptic inhibition in humans, and that genetically encoded impairment of KCC2 function, due to effects on gene dosage, intrinsic activity, or extrinsic regulation, can influence epilepsy phenotypes in patients. Accordingly, KCC2 could be a target for a novel antiepileptic strategies that aims to restore GABA inhibition by facilitating Cl extrusion. Such drugs could have relevance for pharmaco-resistant epilepsies and possibly other diseases characterized by synaptic hyperexcitability, such as the spectrum autism disorders.
© The Author(s) 2016.
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8 MeSH Terms
Integrated compensatory network is activated in the absence of NCC phosphorylation.
Grimm PR, Lazo-Fernandez Y, Delpire E, Wall SM, Dorsey SG, Weinman EJ, Coleman R, Wade JB, Welling PA
(2015) J Clin Invest 125: 2136-50
MeSH Terms: Amiloride, Ammonia, Animals, Biological Transport, Blood Pressure, Carbonic Anhydrases, Chlorides, Disease Models, Animal, Enzyme Activation, Epithelial Sodium Channels, Gene Expression Profiling, Gene Regulatory Networks, Gitelman Syndrome, Ketoglutaric Acids, Kidney Glomerulus, Male, Mice, Mice, Knockout, Natriuresis, Nephrons, Paracrine Communication, Phosphorylation, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Receptors, Notch, Receptors, Purinergic P2, Renal Reabsorption, Signal Transduction, Sodium Chloride, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 3
Show Abstract · Added May 3, 2017
Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.
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31 MeSH Terms
Intrarenal dopamine deficiency leads to hypertension and decreased longevity in mice.
Zhang MZ, Yao B, Wang S, Fan X, Wu G, Yang H, Yin H, Yang S, Harris RC
(2011) J Clin Invest 121: 2845-54
MeSH Terms: Aldosterone, Animals, Aquaporin 2, Aromatic-L-Amino-Acid Decarboxylases, Cyclooxygenase 2, Dopamine, Hypertension, Kidney, Kidney Tubules, Proximal, Longevity, Mice, Mice, Knockout, Receptors, Drug, Renin, Sodium-Bicarbonate Symporters, Sodium-Hydrogen Exchanger 3, Sodium-Hydrogen Exchangers, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 1, Solute Carrier Family 12, Member 3, Symporters
Show Abstract · Added December 10, 2013
In addition to its role as an essential neurotransmitter, dopamine serves important physiologic functions in organs such as the kidney. Although the kidney synthesizes dopamine through the actions of aromatic amino acid decarboxylase (AADC) in the proximal tubule, previous studies have not discriminated between the roles of extrarenal and intrarenal dopamine in the overall regulation of renal function. To address this issue, we generated mice with selective deletion of AADC in the kidney proximal tubules (referred to herein as ptAadc-/- mice), which led to selective decreases in kidney and urinary dopamine. The ptAadc-/- mice exhibited increased expression of nephron sodium transporters, decreased natriuresis and diuresis in response to l-dihydroxyphenylalanine, and decreased medullary COX-2 expression and urinary prostaglandin E2 excretion and developed salt-sensitive hypertension. They had increased renin expression and altered renal Ang II receptor (AT) expression, with increased AT1b and decreased AT2 and Mas expression, associated with increased renal injury in response to Ang II. They also exhibited a substantially shorter life span compared with that of wild-type mice. These results demonstrate the importance of the intrarenal dopaminergic system in salt and water homeostasis and blood pressure control. Decreasing intrarenal dopamine subjects the kidney to unbuffered responses to Ang II and results in the development of hypertension and a dramatic decrease in longevity.
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21 MeSH Terms
AT1A angiotensin receptors in the renal proximal tubule regulate blood pressure.
Gurley SB, Riquier-Brison ADM, Schnermann J, Sparks MA, Allen AM, Haase VH, Snouwaert JN, Le TH, McDonough AA, Koller BH, Coffman TM
(2011) Cell Metab 13: 469-475
MeSH Terms: Animals, Blood Pressure, Hypertension, Kidney Tubules, Proximal, Mice, Receptor, Angiotensin, Type 1, Renin-Angiotensin System, Signal Transduction, Sodium, Sodium-Hydrogen Exchanger 3, Sodium-Hydrogen Exchangers, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 1
Show Abstract · Added August 19, 2013
Hypertension affects more than 1.5 billion people worldwide but the precise cause of elevated blood pressure (BP) cannot be determined in most affected individuals. Nonetheless, blockade of the renin-angiotensin system (RAS) lowers BP in the majority of patients with hypertension. Despite its apparent role in hypertension pathogenesis, the key cellular targets of the RAS that control BP have not been clearly identified. Here we demonstrate that RAS actions in the epithelium of the proximal tubule have a critical and nonredundant role in determining the level of BP. Abrogation of AT(1) angiotensin receptor signaling in the proximal tubule alone is sufficient to lower BP, despite intact vascular responses. Elimination of this pathway reduces proximal fluid reabsorption and alters expression of key sodium transporters, modifying pressure-natriuresis and providing substantial protection against hypertension. Thus, effectively targeting epithelial functions of the proximal tubule of the kidney should be a useful therapeutic strategy in hypertension.
Copyright © 2011 Elsevier Inc. All rights reserved.
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13 MeSH Terms
The Ste20 kinases Ste20-related proline-alanine-rich kinase and oxidative-stress response 1 regulate NKCC1 function in sensory neurons.
Geng Y, Hoke A, Delpire E
(2009) J Biol Chem 284: 14020-8
MeSH Terms: Animals, Calibration, Cell Line, Cell Separation, Ganglia, Spinal, Gene Expression Profiling, Gene Expression Regulation, Gene Knockdown Techniques, Mice, Mice, Knockout, Oxidative Stress, Protein Kinases, Protein-Serine-Threonine Kinases, RNA, Small Interfering, Rats, Reverse Transcriptase Polymerase Chain Reaction, Sensory Receptor Cells, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 2, Thallium
Show Abstract · Added August 13, 2010
NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.
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20 MeSH Terms
NKCC1 and KCC2 prevent hyperexcitability in the mouse hippocampus.
Zhu L, Polley N, Mathews GC, Delpire E
(2008) Epilepsy Res 79: 201-12
MeSH Terms: 4-Aminopyridine, Animals, Blotting, Western, Chlorides, Electrophysiology, Extracellular Space, GABA Agonists, Genotype, Hippocampus, In Vitro Techniques, Mice, Mice, Knockout, Muscimol, Neuroglia, Patch-Clamp Techniques, Potassium Channel Blockers, Pyramidal Cells, Seizures, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 2, Symporters
Show Abstract · Added August 13, 2010
During postnatal development of the central nervous system (CNS), the response of GABA(A) receptors to its agonist undergoes maturation from depolarizing to hyperpolarizing. This switch in polarity is due to the developmental decrease of the intracellular Cl concentration in neurons. Here we show that absence of NKCC1 in P9-P13 CA3 pyramidal neurons, through genetic manipulation or through bumetanide inhibition, results in a significant increase in cell excitability. Furthermore, the pro-convulsant agent 4-aminopyridine induces seizure-like events in NKCC1-null mice but not in wild-type mice. Measurements of muscimol responses in the presence and absence of NKCC1 shows that the Na-K-2Cl cotransporter only marginally affects intracellular Cl(-) in P9-P13 CA3 principal neurons. However, large increases in intracellular Cl(-) are observed in CA3 pyramidal neurons following increased hyperexcitability, indicating that P9-P13 CA3 pyramidal neurons lack robust mechanisms to regulate intracellular Cl(-) during high synaptic activity. This increase in the Cl(-) concentration is network-driven and activity-dependent, as it is blocked by the non-NMDA glutamate receptor antagonist DNQX. We also show that expression of the outward K-Cl cotransporter, KCC2, prevents the development of hyperexcitability, as a reduction of KCC2 expression by half results in increased susceptibility to seizure under control and 4-AP conditions.
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21 MeSH Terms
NKCC1 phosphorylation stimulates neurite growth of injured adult sensory neurons.
Pieraut S, Laurent-Matha V, Sar C, Hubert T, Méchaly I, Hilaire C, Mersel M, Delpire E, Valmier J, Scamps F
(2007) J Neurosci 27: 6751-9
MeSH Terms: Age Factors, Animals, Cells, Cultured, Humans, Intracellular Fluid, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurites, Neurons, Afferent, Phosphorylation, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 2
Show Abstract · Added August 13, 2010
Peripheral nerve section promotes regenerative, elongated neuritic growth of adult sensory neurons. Although the role of chloride homeostasis, through the regulation of ionotropic GABA receptors, in the growth status of immature neurons in the CNS begins to emerge, nothing is known of its role in the regenerative growth of injured adult neurons. To analyze the intracellular Cl- variation after a sciatic nerve section in vivo, gramicidin perforated-patch recordings were used to study muscimol-induced currents in mice dorsal root ganglion neurons isolated from control and axotomized neurons. We show that the reversal potential of muscimol-induced current, E(GABA-A), was shifted toward depolarized potentials in axotomized neurons. This was attributable to Cl- influx because removal of extracellular Cl- prevented this shift. Application of bumetanide, an inhibitor of NKCC1 cotransporter and E(GABA-A) recordings in sensory neurons from NKCC1-/- mice, identified NKCC1 as being responsible for the increase in intracellular Cl- in axotomized neurons. In addition, we demonstrate with a phospho-NKCC1 antibody that nerve injury induces an increase in the phosphorylated form of NKCC1 in dorsal root ganglia that could account for intracellular Cl- accumulation. Time-lapse recordings of the neuritic growth of axotomized neurons show a faster growth velocity compared with control. Bumetanide, the intrathecal injection of NKCC1 small interfering RNA, and the use of NKCC1-/- mice demonstrated that NKCC1 is involved in determining the velocity of elongated growth of axotomized neurons. Our results clearly show that NKCC1-induced increase in intracellular chloride concentration is a major event accompanying peripheral nerve regeneration.
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13 MeSH Terms