NKCC1 Activity Research Articles

Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+exchange

Na(+)-K(+)-2Cl(-) cotransport (NKCC) mediates the movement of two Cl(-) ions for one Na(+) and one K(+) ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl(-) uptake in Xenopus laevis oocytes, as measured using (36)Cl, is twice the value of K(+) uptake, as determined using (86)Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K(+) uptake with only a minimal increase in bumetanide-sensitive Cl(-) uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K(+)/K(+) and Cl(-)/Cl(-) exchange. Although significant stimulation of K(+)/K(+) exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K(+)/K(+) exchange.

The Na–K–Cl cotransporter in the brain edema of acute liver failure

Astrocyte swelling and brain edema associated with increased intracranial pressure are major complications of acute liver failure (ALF). The mechanism for such astrocyte swelling/brain edema, however, is not well understood. We recently found that ammonia, a key etiological factor in ALF, caused the activation of the Na-K-Cl cotransporter-1 (NKCC1) in cultured astrocytes, and that inhibition of such activation led to a reduction in astrocyte swelling, suggesting that NKCC1 activation may be an important factor in the mechanism of brain edema in ALF. To determine whether NKCC activation is also involved in brain edema in vivo, we examined whether NKCC activation occurs in the thioacetamide (TAA) rat model of ALF and determined whether treatment with the NKCC inhibitor bumetanide reduces the severity of brain edema in TAA-treated rats. Brain water content was measured using the gravimetric method. NKCC1 phosphorylation and protein expression were measured by Western blots. NKCC activity was measured in brain cortical slices. NKCC activity was elevated in brain cortical slices of TAA-treated rats as compared to sham animals. Western blot analysis showed significant increases in total as well as phosphorylated (activated) NKCC1 protein expression in the cortical tissue. These findings were associated with a significant increase in brain water content which was attenuated by treatment with the NKCC inhibitor bumetanide. Our studies suggest the involvement of NKCC in the development of brain edema in experimental ALF, and that targeting NKCC may represent a useful therapeutic strategy in humans with ALF.

Stimulation of human and mouse erythrocyte Na+–K+–2Cl− cotransport by osmotic shrinkage does not involve AMP‐activated protein kinase, but is associated with STE20/SPS1‐related proline/alanine‐rich kinase activation

This study was undertaken to investigate whether the mechanism of increased Na(+)-K(+)-2Cl(-) (NKCC1) cotransporter activity by osmotic shrinkage involved AMP-activated protein kinase (AMPK) activation. AMPK was found to phosphorylate a recombinant GST-dogfish (1-260) NKCC1 fragment at Ser38 and Ser214, corresponding to Ser77 and Ser242 in human NKCC1, respectively. Incubation of human erythrocytes with 20 microM A769662 AMPK activator increased Ser242 NKCC1 phosphorylation but did not stimulate (86)Rb(+) uptake. Under hypertonic conditions in human red blood cells (RBCs) incubated with 0.3 M sucrose, NKCC1 activity increased as measured by bumetanide-sensitive (86)Rb(+) uptake and AMPK was activated. However, there was no effect of AMPKalpha1 deletion in mouse RBCs on the increased rate of (86)Rb(+) uptake induced by hyperosmolarity. AMPK activation by osmotic shrinkage of mouse RBCs was abrogated by 10 microM STO-609 CaMKKbeta inhibitor, but incubation with STO-609 did not affect the increase in (86)Rb(+) uptake induced by hyperosmolarity. Osmotic shrinkage of human and mouse RBCs led to activation loop phosphorylation of the STE20/SPS1-related proline/alanine-rich kinase (SPAK) at Thr233, which was accompanied by phosphorylation of NKCC1 at Thr203/207/212, one of which (Thr207) is responsible for cotransporter activation. Therefore, phosphorylation-induced activation of NKCC1 by osmotic shrinkage does not involve AMPK and is likely to be due to SPAK activation.

On the substrate recognition and negative regulation of SPAK, a kinase modulating Na+-K+-2Cl− cotransport activity

Threonines targeted by Ste20-related proline-alanine-rich kinase (SPAK) for phosphorylation have been identified in Na+-K+-2Cl(-) cotransporter type 1 (NKCC1), NKCC2, and Na+-Cl(-) cotransporter (NCC). However, what constitutes the substrate recognition of the kinase is still unknown. Using site-directed mutagenesis and functional measurement of NKCC1 activity in Xenopus laevis oocytes, we determined that SPAK recognizes two threonine residues separated by four amino acids. Addition or removal of a single residue abrogated SPAK activation of NKCC1. Although both threonines are followed by hydrophobic residues, in vivo experiments have determined that SPAK activation of the cotransporter only requires a hydrophobic residue after the first threonine. Interestingly, downstream of the second threonine residue, we have identified a conserved aspartic acid residue which is critical for NKCC1 function. Mouse SPAK activity requires phosphorylation of two specific residues by WNK [with no lysine (K)] kinases: a threonine (T243) in the catalytic domain and a serine (S383) in the regulatory domain. We found that mutating the threonine residue into a glutamic acid (T243E) combined with mutation of the serine into an aspartic acid (S383D) rendered SPAK constitutively active. Surprisingly, alanine substitution of S383 or mutation of residues surrounding this residue also resulted in a constitutively active kinase. Interestingly, deletion of amino acids 356-398 identified another serine residue in the catalytic domain (S321) as another putative target of WNK phosphorylation. We found that WNK4 is capable of stimulating the deletion mutant when S321 is present, but not when S321 is mutated into an alanine.

Multiple Pathways for Protein Phosphatase 1 (PP1) Regulation of Na-K-2Cl Cotransporter (NKCC1) Function: THE N-TERMINAL TAIL OF THE Na-K-2Cl COTRANSPORTER SERVES AS A REGULATORY SCAFFOLD FOR Ste20-RELATED PROLINE/ALANINE-RICH KINASE (SPAK) AND PP1

The Na-K-2Cl cotransporter (NKCC1) participates in epithelial transport and in cell volume maintenance by mediating the movement of ions and water across plasma membranes. Functional studies have previously demonstrated that NKCC1 activity is stimulated by protein phosphatase 1 (PP1) inhibitors. In this study, we utilized both in vivo (heterologous cRNA expression in Xenopus laevis oocytes) and in vitro ((32)P-phosphorylation assays with glutathione S-transferase fusion proteins) experiments to determine whether PP1 exerts its inhibitory effect directly on the cotransporter, or indirectly by affecting the activating kinase. We found that PP1 reduced NKCC1 activity in oocytes under both isotonic and hypertonic conditions to the same level as in water-injected controls. Interestingly, mutation of key residues in the PP1 binding motif located in the N-terminal tail of NKCC1 significantly reduced the inhibitory effect of PP1. In vitro experiments performed with recombinant PP1, SPAK (Ste20-related proline/alanine-rich kinase, which activates NKCC1), and the N terminus of NKCC1 fused to glutathione S-transferase demonstrated that PP1 dephosphorylated both the kinase and the cotransporter in a time-dependent manner. More importantly, PP1 dephosphorylation of SPAK was significantly greater when protein-protein interaction between the kinase and the N-terminal tail of NKCC1 was present in the reaction, indicating the necessity of scaffolding the phosphatase and kinase in proximity to one another. Taken together, our data are consistent with PP1 inhibiting NKCC1 activity directly by dephosphorylating the cotransporter and indirectly by dephosphorylating SPAK.

Modulation of NaCl absorption by [HCO(3)(-)] in the marine teleost intestine is mediated by soluble adenylyl cyclase.

Intestinal HCO(3)(-) secretion and NaCl absorption are essential for counteracting dehydration in marine teleost fish. We investigated how these two processes are coordinated in toadfish. HCO(3)(-) stimulated a luminal positive short-circuit current (I(sc)) in intestine mounted in Ussing chamber, bathed with the same saline solution on the external and internal sides of the epithelium. The I(sc) increased proportionally to the [HCO(3)(-)] in the bath up to 80 mM NaHCO(3), and it did not occur when NaHCO(3) was replaced with Na(+)-gluconate or with NaHCO(3) in Cl(-)-free saline. HCO(3)(-) (20 mM) induced a approximately 2.5-fold stimulation of I(sc), and this [HCO(3)(-)] was used in all subsequent experiments. The HCO(3)(-)-stimulated I(sc) was prevented or abolished by apical application of 10 muM bumetanide (a specific inhibitor of NKCC) and by 30 microM 4-catechol estrogen [CE; an inhibitor of soluble adenylyl cyclase (sAC)]. The inhibitory effects of bumetanide and CE were not additive. The HCO(3)(-)-stimulated I(sc) was prevented by apical bafilomycin (1 microM) and etoxolamide (1 mM), indicating involvement of V-H(+)-ATPase and carbonic anhydrases, respectively. Immunohistochemistry and Western blot analysis confirmed the presence of an NKCC2-like protein in the apical membrane and subapical area of epithelial intestinal cells, of Na(+)/K(+)-ATPase in basolateral membranes, and of an sAC-like protein in the cytoplasm. We propose that sAC regulates NKCC activity in response to luminal HCO(3)(-), and that V-H(+)-ATPase and intracellular carbonic anhydrase are essential for transducing luminal HCO(3)(-) into the cell by CO(2)/HCO(3)(-) hydration/dehydration. This mechanism putatively coordinates HCO(3)(-) secretion with NaCl and water absorption in toadfish intestine.

Promoter hypomethylation upregulates Na +–K +–2Cl − cotransporter 1 in spontaneously hypertensive rats

The Na +–K +–2Cl − cotransporter 1 (NKCC1) is one of several transporters that have been implicated for development of hypertension since NKCC1 activity is elevated in hypertensive aorta and vascular contractions are inhibited by bumetanide, an inhibitor of NKCC1. We hypothesized that promoter hypomethylation upregulates the NKCC1 in spontaneously hypertensive rats (SHR). Thoracic aortae and mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The expression levels of nkcc1 mRNA and protein in aortae and heart tissues were measured by real-time PCR and Western blot, respectively. The methylation status of nkcc1 promoter region was analyzed by combined bisulfite restriction assay (COBRA) and bisulfite sequencing. Phenylephrine-induced vascular contraction in a dose-dependent manner, which was inhibited by bumetanide. The inhibition of dose–response curves by bumetanide was much greater in SHR than in Wistar Kyoto (WKY) normotensive rats. The expression levels of nkcc1 mRNA and of NKCC1 protein in aortae and heart tissues were higher in SHR than in WKY. Nkcc1 gene promoter was hypomethylated in aortae and heart than those of WKY. These results suggest that promoter hypomethylation upregulates the NKCC1 expression in aortae and heart of SHR.

Role of SPAK binding sites in SLC12 cotransporters basal activity and regulation by WNK3

With‐no‐lysine kinase 3 (WNK3) is a powerful activator of NKCC1/2 and NCC while is an inhibitor of KCC1 ‐ KCC4. WNKs lie upstream of the STE‐20 kinases SPAK/OSR1. It is known that interaction with SPAK occurs through the SPAK‐binding motif, RFx(V/I). WNK3 contains three such motifs, NCC and NKCC2 one; while KCC4 lacks this motif. In the present study we examined the relevance of SPAK binding sites for NCC, NKCC2, and KCC4 basal activity and regulation by WNK3. The sites of WNK3 (F242A, F745A, or F1337A; one at a time), NCC (F19A), and NKCC2 (F17A) were eliminated. Basal activity and effect of WNK3 was assessed by measuring the tracer 22Na+ or 86Rb+uptake in Xenopus oocytes injected with wild type or mutant cRNAs. Basal activity of NCC‐F19A and NKCC2‐F17A was reduced by ~50%. However, the response to WNK3 and to intracellular chloride depletion was still present. The effect of WNK3 toward NKCC2, but not NCC or KCC4, was eliminated by WNK3‐F1337A. Interestingly, the WNK3 effect towards NCC or KCC4, but not NKCC2, was prevented in WNK3‐F242A. Biochemical analysis revealed that catalytic activity is preserved in both, WNK3‐F242A and WNK3‐F1337A. Thus, we conclude that basal activity of NCC and NKCC2, but not KCC4 requires the presence of an SPAK binding site on the cotransporter. Modulation of NCC, NKCC2, and KCC4 by WNK3 requires SPAK binding sites in the kinase, however, different sites are used for regulation of NKCC2 in contrast to NCC or KCC4.

ON THE SUBSTRATE RECOGNITION AND AUTOINHIBITORY DOMAIN OF SPAK

Threonines targeted by SPAK for phosphorylation have been identified in NKCC1, NKCC2, and NCC. However, what constitutes the substrate recognition of the kinase is still unknown. Using site‐directed mutagenesis and functional measurement of NKCC1 activity in Xenopus laevis oocytes, we determined that SPAK recognizes two threonine residues separated by four amino acids. Addition or removal of a single residue abrogated SPAK activation of NKCC1. Although both threonines are followed by hydrophobic residues, only the first residue is essential for SPAK activation of the cotransporter. Negatively charged residues around the sites targeted for phosphorylation by other kinases have been shown to be involved in substrate recognition. Thus, we mutated two aspartate residues flanking the two threonines. Whereas alanine mutation of the upstream residue had no effect, alanine substitution of the downstream residue inhibited cotransporter activity. However, conserved substitution of this residue into glutamate preserved NKCC1 function. SPAK activity requires phosphorylation of two residues by WNK kinases. We found that mutating T243E combined with S383D rendered the kinase constitutively active. Surprisingly, S383A mutation or mutation of residues surrounding this residue also resulted in an active kinase. These data indicate the possibility of an inhibitory domain which is affected by WNK phosphorylation.

Heterologous regulation of anion transporters by menthol in human airway epithelial cells

The present study concerns previously unreported effects of menthol, a cyclic terpene alcohol produced by the peppermint herb, on anion transporters in polarized human airway Calu-3 epithelia. Application of menthol (0.01–1 mM) attenuated transepithelial anion transport, estimated as short-circuit currents ( I SC), after stimulation by forskolin (10 µM) but not before. In contrast, menthol potentiated forskolin-stimulated and -unstimulated apical Cl − conductance, which reflected the cystic fibrosis transmembrane conductance regulator (CFTR: the cAMP-regulated Cl − channel)-mediated conductance, without correlation to changes in cytosolic cAMP levels. These results indicate that menthol-induced attenuation of forskolin-induced I SC despite CFTR up-regulation was due to cAMP-independent inhibition of basolateral anion uptake, which is the rate-limiting step for transepithelial anion transport. Analyses of the responsible basolateral anion transporters revealed that forskolin increased both bumetanide (an inhibitor of the basolateral Na +–K +–2Cl − cotransporter [NKCC1])- and DNDS (an inhibitor of basolateral HCO 3 −-dependent anion transporters [NBC1/AE2])-sensitive I SC in the control whereas only the former was prevented by the application of menthol. Neither the bumetanide- nor DNDS-sensitive component was, however, reduced by menthol without forskolin. These heterologous effects of menthol were reproduced by latrunculin B, an inhibitor of actin polymerization. F-actin staining showed that menthol prevented forskolin-stimulated rearrangements of actin microfilaments without affecting the distribution of forskolin-unstimulated microfilaments. Collectively, menthol functions as an activator of CFTR and prevents activation of NKCC1 without affecting NBC1/AE although all of these transporters are commonly cAMP-dependent. The heterologous effects may be mediated by the actin cytoskeleton, which interacts with CFTR and NKCC1.

MOLECULAR DETERMINANT(S) OF HYPER‐OSMOTICALLY ACTIVATED NKCC1‐MEDIATED K+/K+ EXCHANGE

Na+‐K+‐2Cl− cotransport mediates the movement of two Cl− ions for one Na+ and one K+ ion. Under isosmotic conditions or SPAK/WNK4 activation, the NKCC1‐mediated bumetanide‐sensitive Cl− uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. In contrast, under hyperosmotic conditions, there is a very significant activation of the bumetanide‐sensitive K+ uptake with only a minimal increase in Cl− uptake. This indicates that when stimulated by hypertonicity, the cotransporter mediates Na+‐K+‐2Cl− cotransport as well as K+/K+ exchange. Interestingly, this stimulation of K+/K+ exchange was only observed with NKCC1, but not NKCC2. In order to identify the molecular determinant(s) of this NKCC1‐specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2, nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the second extracellular loop between membrane spanning domains three and four is involved in the hyperosmotic stimulation of K+ uptake (K+/K+ exchange) in NKCC1. However, replacing only this loop in NKCC2 was insufficient for K+ uptake activation, suggesting an interaction between this loop and a yet to be identified region of the cotransporter.

Skeletal Muscle Regulatory Responses to Increased Extracellular Lactate

Mammalian skeletal muscle cells exhibit cell shrinkage followed by regulatory volume increase (RVI) in response to increases in extracellular osmolarity caused by addition of concentrated NaCl or sucrose. We hypothesized that increased extracellular sodium lactate would similarly result in a transient loss of cell volume, followed by RVI, and that the RVI would be more rapid and pronounced than that seen with NaCl due to entry of lactate into the cell. The present study measured the time course of change single fibre width in response to varied increases in extracellular NaCl or Na‐lactate. Adult mouse EDL single fibres were isolated using collagenase and incubated in DMEM prior to and during experimentation. Fibre images were obtained at 3–6 s intervals for up to 40 min. Fibre images were analyzed for width at 2 sites. Increased osmolarity resulted in a rapid decrease in fibre width, followed by RVI. The volume recovery was faster and more complete when [lactate] ≥20 mmol/L. There was a linear relationship between increases extracellular [lactate], cell shrinkage, and magnitude of RVI. It appears that the inward transport of lactate into skeletal muscle contributes, together with increased NKCC activity, to the RVI in mammalian skeletal muscle.Supported by NSERC of Canada.

Mutation of the Na+-K+-2Cl−cotransporter NKCC2 in mice is associated with severe polyuria and a urea-selective concentrating defect without hyperreninemia

The bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter NKCC2, located in the thick ascending limb of Henle's loop, plays a critical role in the kidney's ability to concentrate urine. In humans, loss-of-function mutations of the solute carrier family 12 member 1 gene (SLC12A1), coding for NKCC2, cause type I Bartter syndrome, which is characterized by prenatal onset of a severe polyuria, salt-wasting tubulopathy, and hyperreninemia. In this study, we describe a novel chemically induced, recessive mutant mouse line termed Slc12a1(I299F) exhibiting late-onset manifestation of type I Bartter syndrome. Homozygous mutant mice are viable and exhibit severe polyuria, metabolic alkalosis, marked increase in plasma urea but close to normal creatininemia, hypermagnesemia, hyperprostaglandinuria, hypotension,, and osteopenia. Fractional excretion of urea is markedly decreased. In addition, calcium and magnesium excretions are more than doubled compared with wild-type mice, while uric acid excretion is twofold lower. In contrast to hyperreninemia present in human disease, plasma renin concentration in homozygotes is not increased. The polyuria observed in homozygotes may be due to the combination of two additive factors, a decrease in activity of mutant NKCC2 and an increase in medullary blood flow, due to prostaglandin-induced vasodilation, that impairs countercurrent exchange of urea in the medulla. In conclusion, this novel viable mouse line with a missense Slc12a1 mutation exhibits most of the features of type I Bartter syndrome and may represent a new model for the study of this human disease.

NKCC1 and hypertension: a novel therapeutic target involved in the regulation of vascular tone and renal function

The present review summarizes recent advances in our understanding of the mechanisms involving the housekeeping Na+, K+, 2Cl(-) cotransporter (NKCC1) in blood pressure (BP) regulation. High-ceiling diuretics (HCDs), known potent inhibitors of NKCC1, renal-specific NKCC2 and four isoforms of K+, Cl(-) cotransporters decrease [Cl(-)]i, hyperpolarize vascular smooth muscle cells and suppress myogenic tone and contractions evoked by modest depolarization, phenylephrine, angiotensin II and uridine triphosphate. These actions are absent in NKCC1(-/-) mice, indicating that HCDs interact with NKCC1 rather than with other potential targets. NKCC1-null mice have decreased baseline BP but exhibit augmented BP increment evoked by high-salt diets. NKCC1 deficiency causes approximately three-fold elevation in plasma renin concentrations and attenuates HCD-induced renin production. In addition to HCDs, NKCC1 is also inhibited by extracellular HCO3(-) in the range corresponding to its concentration in ischemic extracellular fluids. NKCC1 modulates BP through vascular and renal effects. In addition to BP regulation, the decreased baseline activity of this carrier or its suppression by chronic treatment with HCDs may lead to inhibition of myogenic tone and progression of end-stage renal disease. NKCC1 activation in ischemia-induced acidosis may contribute to stroke via glutamate release caused by astrocyte swelling.

The Na–K–Cl Co-transporter in astrocyte swelling

Ion channels, exchangers and transporters are known to be involved in cell volume regulation. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na(+)-K(+)-Cl(-) cotransporters has been shown to regulate cell volume in many conditions. The Na(+)-K(+)-Cl- cotransporters (NKCC) are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. Studies have established the role of NKCC1 in astrocyte swelling/brain edema in ischemia and trauma. Our recent studies suggest that NKCC1 activation is also involved in astrocyte swelling induced by ammonia and in the brain edema in the thioacetamide model of acute liver failure. This review will focus on mechanisms of NKCC1 activation and its contribution to astrocyte swelling/brain edema in neurological disorders, with particular emphasis on ammonia neurotoxicity and acute liver failure.

Decreased NKCC1 Activity in Erythrocytes From African Americans With Hypertension and Dyslipidemia

Recent studies demonstrated a key role of ubiquitous isoform of Na+,K+,2Cl- co-transport (NKCC1) in regulation of myogenic tone and peripheral resistance. We examined the impact of race, gender, and plasma lipid on NKCC1 activity in French Canadians and African Americans with hypertension and dyslipidemia. NKCC and passive erythrocyte membrane permeability to K+, measured as ouabain-resistant, bumetanide-sensitive, and (ouabain+bumetanide)-resistant 86Rb influx, respectively, were compared in 111 French-Canadian men, 107 French-Canadian women, 26 African-American men, and 45 African-American women with essential hypertension and dyslipidemia. The African-American men and women were 7 years younger and presented twofold decreased plasma triglycerides compared to their French-Canadian counterparts (P < 0.01) whereas body mass index (BMI), total cholesterol, low-density lipoprotein, and high-density lipoprotein (HDL) were not different. NKCC was respectively 50 and 38% lower in the African-American men and women than in the French Canadians (P < 0.005) without any differences in passive erythrocyte membrane permeability for K+. We did not observe any impact of age on NKCC in all groups under investigation, whereas plasma triglycerides correlated positively with the activity of this carrier in the French-Canadian men only. NKCC1 activity is lower in erythrocytes of African Americans with essential hypertension and dyslipidemia than in Caucasian counterparts. We suggest that decreased NKCC1 may contribute to the feature of the pathogenesis of salt-sensitive hypertension seen in African Americans.

A possible catalytic role for NH4+in Na+reabsorption across the thick ascending limb

A possible catalytic role for NH₄⁺in Na⁺reabsorption across the thick ascending limb

Protein aggregation in neurons following OGD: a role for Na+ and Ca2+ ionic dysregulation

In this study, we investigated whether disruption of Na(+) and Ca(2+) homeostasis via activation of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchange (NCX(rev)) affects protein aggregation and degradation following oxygen-glucose deprivation (OGD). Cultured cortical neurons were subjected to 2 h OGD and 1-24 h reoxygenation (REOX). Redistribution of ubiquitin and formation of ubiquitin-conjugated protein aggregates occurred in neurons as early as 2 h REOX. The protein aggregation progressed further by 8 h REOX. There was no significant recovery at 24 h REOX. Moreover, the proteasome activity in neurons was inhibited by 80-90% during 2-8 h REOX and recovered partially at 24 h REOX. Interestingly, pharmacological inhibition or genetic ablation of NKCC1 activity significantly decreased accumulation of ubiquitin-conjugated protein aggregates and improved proteasome activity. A similar protective effect was obtained by blocking NCX(rev) activity. Inhibition of NKCC1 activity also preserved intracellular ATP and Na(+) homeostasis during 0-24 h REOX. In a positive control study, disruption of endoplasmic reticulum Ca(2+) with thapsigargin triggered redistribution of free ubiquitin and protein aggregation. We conclude that overstimulation of NKCC1 and NCX(rev) following OGD/REOX partially contributes to protein aggregation and proteasome dysfunction as a result of ionic dysregulation.

Tracing the origin of different red blood cell NKCC activation modes within birds

Tracing the origin of different red blood cell NKCC activation modes within birds

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

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.