Pacheco Álvarez, Diana
Thumbnail Image
Preferred name
Pacheco Álvarez, Diana
Official Name
Pacheco Álvarez, Diana
Alternative Name
dpacheco
Main Affiliation
ORCID
Scopus Author ID
14827257500
Researcher ID
DVP-2845-2022

Research Output

Filters

6
6
6
Reset filters

Settings
Now showing 1 - 6 of 6
No Thumbnail Available
Publication

WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family

2012 , Pacheco Álvarez, Diana , Vázquez, Norma , Castañeda-Bueno, María , De los Heros, Paola , Cortes-González, César , Moreno, Erika , Meade, Patricia , Bobadilla, Norma A. , Gamba, Gerardo

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.

View
No Thumbnail Available
Publication

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity

2018 , Murillo-de-Ozores, Adrián Rafael , Rodríguez Moya, Alejandro , Bazúa-Valenti, Silvana , Leyva Ríos, Karla , Vázquez, Norma , Pacheco Álvarez, Diana , Rosa-Velázquez, Inti A. de la , Wengi, Agnieszka , Stone, Kathryn L. , Junhui, Zhang , Loffing, Johannes , Lifton, Richard P. , Chao-Ling , Yang , Ellison, David H. , Gamba, Gerardo , Castañeda-Bueno, María

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate. © 2018 American Society for Biochemistry and Molecular Biology Inc. All rights reserved.

View
No Thumbnail Available
Publication

The European Eel NCCβ Gene Encodes a Thiazide-resistant Na-Cl Cotransporter

2016 , Moreno, Erika , Plata, Consuelo , Rodríguez-Gama, Alejandro , Argaiz, Eduardo R. , Vázquez, Norma , Leyva Ríos, Karla , Islas, León , Cutler, Christopher , Pacheco Álvarez, Diana , Mercado, Adriana , Cariño-Cortés, Raquel , Castañeda-Bueno, María , Gamba, Gerardo

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter. © 2016 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.

View
No Thumbnail Available
Publication

WNK-SPAK-NCC cascade revisited: WNK1 stimulates the activity of the Na-Cl cotransporter via SPAK, an effect antagonized by WNK4

2014 , Chávez-Canales, María , Zhang, Chong , Soukaseum, Christelle , Moreno, Erika , Pacheco Álvarez, Diana , Vidal-Petiot, Emmanuelle , Castañeda-Bueno, María , Vázquez, Norma , Rojas-Vega, Lorena , Meermeier, Nicholas P. , Rogers, ,Shaunessy , Jeunemaitre, Xavier , Yang, Chao-Ling , Ellison, David H. , Gamba, Gerardo , Hadchouel, Juliette

The with-no-lysine (K) kinases, WNK1 and WNK4, are key regulators of blood pressure. Their mutations lead to familial hyperkalemic hypertension (FHHt), associated with an activation of the Na-Cl cotransporter (NCC). Although it is clear that WNK4 mutants activate NCC via Ste20 proline-alanine-rich kinase, the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice (WNK1(+/FHHt)) with WNK4(-/-) mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1(+/FHHt) mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and Ste20 proline-alanine-rich kinase-dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC. © 2014 American Heart Association, Inc.

View
No Thumbnail Available
Publication

WNK3 and WNK4 exhibit opposite sensitivity with respect to cell volume and intracellular chloride concentration

2020 , Pacheco Álvarez, Diana , Carrillo-Pérez, Diego Luis , Mercado, Adriana , Leyva Ríos, Karla , Moreno, Erika , Castañeda-Bueno, María , Elisa Hernández-Mercado , Vázquez, Norma , Gamba, Gerardo

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK’s carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC. Copyright © 2020 the American Physiological Society

View
No Thumbnail Available
Publication

A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters

2010 , Castañeda-Bueno, María , Vázquez, Norma , Bustos-Jaimes, Ismael , Hernández, Damian , Rodríguez-Lobato, Erika , Pacheco Álvarez, Diana , Cariño-Cortés, Raquel , Moreno, Erika , Bobadilla, Norma A. , Gamba, Gerardo

Little is known about the residues that control the binding and affinity of thiazide-type diuretics for their protein target, the renal Na(+)-Cl(-) cotransporter (NCC). Previous studies from our group have shown that affinity for thiazides is higher in rat (rNCC) than in flounder (flNCC) and that the transmembrane region (TM) 8-12 contains the residues that produce this difference. Here, an alignment analysis of TM 8-12 revealed that there are only six nonconservative variations between flNCC and mammalian NCC. Two are located in TM9, three in TM11, and one in TM12. We used site-directed mutagenesis to generate rNCC containing flNCC residues, and thiazide affinity was assessed using Xenopus laevis oocytes. Wild-type or mutant NCC activity was measured using (22)Na(+) uptake in the presence of increasing concentrations of metolazone. Mutations in TM11 conferred rNCC an flNCC-like affinity, which was caused mostly by the substitution of a single residue, S575C. Supporting this observation, the substitution C576S conferred to flNCC an rNCC-like affinity. Interestingly, the S575C mutation also rendered rNCC more active. Substitution of S575 in rNCC for other residues, such as alanine, aspartate, and lysine, did not alter metolazone affinity, suggesting that reduced affinity in flNCC is due specifically to the presence of a cysteine. We conclude that the difference in metolazone affinity between rat and flounder NCC is caused mainly by a single residue and that this position in the protein is important for determining its functional properties. © American Journal of Physiology-Renal Physiology

View