Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases

Ponce‐Coria, José; San‐Cristobal, Pedro; Kahle, Kristopher T.; Vázquez, Norma; Pacheco‐Alvarez, Diana; Heros, Paola de los; Juàrez, Patricia; Muñoz, Eva; Michel, Gabriela; Bobadilla, Norma A.; Gíménez, Ignacio; Lifton, Richard P.; Hebert, Steven C.; Gamba, Gerardo

doi:10.1073/pnas.0802966105

Cited by 196 publications

(252 citation statements)

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“…The kinase WNK3 is absent from ORNs. Among the four WNK kinases, WNK3 shows the highest expression in the brain, and WNK3 is thought to act as a Cl − sensor involved in protecting cells from excessive Cl − accumulation (45)(46)(47). The absence of WNK3 from ORNs, the simple thermodynamic basis of NKCC1 activity seen in our transport assay, and the absence of the main neuronal Cl − exporter KCC2 (2) all illustrate that these neurons dispense with means for intracellular Cl − reduction and instead optimize Cl − accumulation.…”

Section: Discussionmentioning

confidence: 99%

Molecular components of signal amplification in olfactory sensory cilia

Hengl

Kaneko

Dauner

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The mammalian olfactory system detects an unlimited variety of odorants with a limited set of odorant receptors. To cope with the complexity of the odor world, each odorant receptor must detect many different odorants. The demand for low odor selectivity creates problems for the transduction process: the initial transduction step, the synthesis of the second messenger cAMP, operates with low efficiency, mainly because odorants bind only briefly to their receptors. Sensory cilia of olfactory receptor neurons have developed an unusual solution to this problem. They accumulate chloride ions at rest and discharge a chloride current upon odor detection. This chloride current amplifies the receptor potential and promotes electrical excitation. We have studied this amplification process by examining identity, subcellular localization, and regulation of its molecular components. We found that the Na + /K + /2Cl − cotransporter NKCC1 is expressed in the ciliary membrane, where it mediates chloride accumulation into the ciliary lumen. Gene silencing experiments revealed that the activity of this transporter depends on the kinases SPAK and OSR1, which are enriched in the cilia together with their own activating kinases, WNK1 and WNK4. A second Cl − transporter, the Cl − /HCO 3 − exchanger SLC4A1, is expressed in the cilia and may support Cl − accumulation. The calcium-dependent chloride channel TMEM16B (ANO2) provides a ciliary pathway for the excitatory chloride current. These findings describe a specific set of ciliary proteins involved in anion-based signal amplification. They provide a molecular concept for the unique strategy that allows olfactory sensory neurons to operate as efficient transducers of weak sensory stimuli.chloride | olfaction | sensory transduction | transport | kinase M ammalian olfactory receptor neurons (ORNs) present to the air a tuft of sensory cilia equipped with odorant receptors. Upon contact with odorants, these receptors actuate a transduction cascade that leads to firing of action potentials. This cascade has an unusual, two-stage organization (1). First, the activated odorant receptors induce a rise of the second messengers cAMP and Ca 2+ in the cilia, a process that involves cAMP-gated, Ca 2+ -permeable ion channels. In the second stage, inflowing Ca 2+ opens Cl − channels. By conducting a depolarizing Cl − efflux from the cilia, these channels amplify the receptor potential approximately 10-fold, thus helping to excite the neuron even when stimulation is weak. ORNs accumulate chloride through the Na + /K + /2Cl − cotransporter NKCC1 and maintain an elevated intracellular Cl − concentration (2, 3) to support amplification. Accordingly, gene ablation of NKCC1, as well as the pharmacologic suppression of Cl − accumulation or Cl − efflux, strongly inhibits the sensory response of ORNs (3-5). Although these observations provide a robust concept for signal amplification, several points are still unclear. These concern both the Cl − accumulation process and the excitatory Cl − currents. First, ...

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Section: Discussionmentioning

confidence: 99%

Molecular components of signal amplification in olfactory sensory cilia

Hengl

Kaneko

Dauner

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…We have shown using WNK3-18a that the D294A substitution not only prevents the positive or negative effects of WNK3 on NKCCs or KCCs, respectively, but completely reverses these effects of WNK3 (2,9,20,21). In the present study, we assessed the consequences of eliminating the catalytic activity in all WNK3 isoforms on their effects on the cotransporters.…”

Section: Effects Of the Catalytically Inactive Wnk3 Variants On The Cmentioning

confidence: 99%

“…10). WNK3 activates NKCCs by increasing their phosphorylation, even when cells are incubated in hypotonic medium, where normally they are inactivated (9,10,20,21). In contrast, the coexpression of KCCs with WNK3 completely inhibits KCC function, even when cells are exposed to a hypotonic medium, in which the KCCs are normally active and dephosphorylated (2,22).…”

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confidence: 96%

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Similar effects of all WNK3 variants on SLC12 cotransporters

Cruz-Rangel

Melo

Vázquez

et al. 2011

American Journal of Physiology-Cell Physiology

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kinase 3 (WNK3) is a member of a subfamily of serine/threonine kinases that modulate the activity of the electroneutral cation-coupled chloride cotransporters. WNK3 activates NKCC1/2 and NCC and inhibits the KCCs. Four splice variants are generated from the WNK3 gene. Our previous studies focused on the WNK3-18a variant. However, it has been suggested that other variants could have different effects on the cotransporters. Thus, the present study was designed to define the effects of all WNK3 variants on members of the SLC12 family. By RT-PCR from a fetal brain library, exons 18b and 22 were separately amplified and subcloned into the original WNK3-18a or catalytically inactive WNK3-D294A to obtain all four potential combinations with and without catalytic activity (18a, 18aϩ22, 18b, and 18bϩ22). The basal activity of the cotransporters and the effects of WNK3 isoforms were assessed in Xenopus laevis oocytes coinjected with each of the WNK3 variant cRNAs. In isotonic conditions, the basal activity of NCC and NKCC1/2 were increased by coinjection with any of the WNK3. The positive effects occurred even in hypotonic conditions, in which the basal activity of NKCC1 is completely prevented. Consistent with these observations, when expressed in hypotonicity, all KCCs were active, but in the presence of any of the WNK3 variants, KCC activity was completely reduced. That is, NKCC1/2 and NCC were inhibited, even in hypertonicity, while KCCs were activated, even in isotonic conditions. We conclude that the effects of all WNK3 variants toward SLC12 proteins are similar.

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“…Commonly, WNK3 may affect the function of NCC in the following two ways. On one hand, WNK3 can regulate the expression of NCC on the cell surface; and on the other hand it may also influence the function of NCC on the cell membrane by phosphorylation (Ponce-Coria et al, 2008). Our observation after immunofluorescence staining showed that normally NCC was mainly distributed on the Cos-7 cell membrane, with a small amount distributed in the cytoplasm ( Figure S2 in Supporting Information).…”

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confidence: 71%

WNK3 interacts with NCC

Wang

Pan

et al. 2016

Sci. China Life Sci.

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Dear Editor, NCC (Na-Cl cotransporter) is a cotransporter mainly distributed in the distal tubule of the kidney, functioning to reabsorb sodium and chloride ions from the tubular fluid into the cells of the renal distal convoluted tubule. It is a transmembrane protein belonging to the SLC12 cotransporter family of electro-neutral cation-coupled chloride cotransporters, which is closely related to hypertension (Gamba, 2005). A loss of NCC function can cause Gitelman syndrome, a disease characterized by low blood pressure, hypocalciuria, hypokalemic metabolic alkalosis, and hypomagnesemia, etc. WNK3 (With No Lysine K 3) is highly expressed in the whole length of the renal tubules (Glover et al., 2009). It was previously found to enhance the activity of NCC in Xenopus oocyte (Rinehart et al., 2005). Exploring the regulatory effect of WNK3 on NCC may be helpful to understand the occurrence of abnormal renal sodium transporters and provide new targets for the treatment of hypertension. However, whether WNK3 interacts directly with NCC in mammalian cells still remains unclear. In this article, we report the results of our co-immunoprecipitation experiments and confocal observations, confirming that WNK3 does interact with NCC and that the kinase domain of WNK3 plays an important role in the regulation of NCC.To explore whether there is reaction between WNK3 and NCC protein, Cos-7 cells were transfected by GFP-NCC with or without HA-WNK3 and co-immunoprecipitation experiments were performed 48 hours after transfection. As shown in Figure 1, anti-HA antibody with the presence of WNK3 immunoprecipitated NCC whereas serum with the absence of WNK3 did not, suggesting that the WNK3 protein interacts with NCC directly.In order to investigate the effect of WNK3 on NCC, HA-NCC was transfected into Cos-7 cells while increasing doses of HA-WNK3. Western-blot was carried out with anti-HA or anti-GAPDH antibodies 48 h later. The NCC protein expression level gradually increased with the increase of WNK3 dosage ( Figure S1 in Supporting Information). The expression of NCC and WNK3 was checked under a confocal fluorescent microscope ( Figure S2 in Supporting Information). The results showed that WNK3 increased NCC expression both on the cell membrane and in the cytoplasm. It has been confirmed that the enzyme region, the amino terminal, and/or the carboxyl terminal are the key elements of kinase activity. However, whether the enzyme region of WNK3 is necessary in regulating NCC is still unclear. We observed the changes of NCC expression after various WNK3 fragments were transferred into the cells. As shown in Figure S3 in Supporting Information, only the WNK3 fragments containing the enzyme region were able to increase the expression of NCC.WNK kinases belong to a subfamily of serine/threonine kinases. It was reported that the activity of NCC could be down-regulated by WNK4 (Moniz et al., 2010). Our study showed that in the mammalian cells WNK3 was also a chloride sensitive kinase to NCC expression, with its effect being the opposit...

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Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases

Cited by 196 publications

References 43 publications

Molecular components of signal amplification in olfactory sensory cilia

Molecular components of signal amplification in olfactory sensory cilia

Similar effects of all WNK3 variants on SLC12 cotransporters

WNK3 interacts with NCC

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