Pacheco Álvarez, Diana

Main Affiliation

Facultad de Ciencias de la Salud - CampCM

Preferred name

Official Name

Pacheco Álvarez, Diana

ORCID

0000-0001-5454-9267

Researcher ID

DVP-2845-2022

Scopus Author ID

14827257500

Now showing 1 - 10 of 15

Some of the metrics are blocked by your
consent settings
A CRAC-like motif in BAX sequence: Relationship with protein insertion and pore activity in liposomes
(2011)
Martínez-Abundis, Eduardo
;
Correa, Francisco
;
Rodríguez, Emma
;
Soria-Castro, Elizabeth
;
Rodríguez-Zavala, José S.
Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein. Copyright © 2011 Elsevier B.V. All rights reserved. © Biochimica et Biophysica Acta (BBA) - Biomembranes
2 7Scopus© Citations 15
Some of the metrics are blocked by your
consent settings
Role of WNK Kinases in the Modulation of Cell Volume
(2018)
Pacheco Álvarez, Diana
;
Gamba, Gerardo
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.
Scopus© Citations 31 2 6
Some of the metrics are blocked by your
consent settings
Angiotensin II signaling increases activity of the renal Na-Cl cotransporter through a WNK4-SPAK-dependent pathway
(2009)
San-Cristóbal, Pedro
;
Pacheco Álvarez, Diana
;
Richardson, Ciaran
;
Ring, Aaron M.
;
Vazquez, Norma
Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K(+) levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K(+) secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K(+) channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (AngII) levels, AngII signaling might modulate WNK4 activity. We show that AngII signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AngII, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by AngII signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. AngII's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by AngII signaling. These effects extend to mammalian cells. AngII increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AngII and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K(+) secretion. © Proceedings of the National Academy of Sciences
Scopus© Citations 216 1 3
Some of the metrics are blocked by your
consent settings
Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases
(2008)
Ponce-Coria, José
;
San-Cristóbal, Pedro
;
Kahle, Kristopher T.
;
Vázquez, Norma
;
Pacheco Álvarez, Diana
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.
2 5Scopus© Citations 208
Some of the metrics are blocked by your
consent settings
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
;
Cortes-González, César
;
Moreno, Erika
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.
Scopus© Citations 30 1 8
Some of the metrics are blocked by your
consent settings
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
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.
1 19Scopus© Citations 13
Some of the metrics are blocked by your
consent settings
Aspectos estructurales, funcionales y patológicos del cotransportador de NaCl sensible a tiazidas
(Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 2014-11)
Moreno, Erika
;
Pacheco Álvarez, Diana
;
Ríos Argaiz, Eduardo
NCC cotransporter is the mayor pathway for sodium chloride reabsorption in the distal nephron and the target of thiazide diuretics which, given their clinical utility in the management of arterial hypertension, are amongst the top sold drugs in the world. NCC protein is of great physiological importance given its role in the maintenance of water and salt homeostasis on the organism. Inactivating mutations in the gene that codes for NCC cause Gitelman's syndrome: an autosomal recessive disease associated with arterial hypotension, metabolic alkalosis, hipokalemia and hypocalciuria. This syndrome represents strong evidence of the relevance of the role of NCC in blood pressure regulation, electrolyte and acid base balance. In this work we review the up to date knowledge regarding this cotransporter with special attention to the molecular aspects of the protein that determine is physiological function and pathological roles. ©Revista de Investigación Clínica
Scopus© Citations 1 1 23
Some of the metrics are blocked by your
consent settings
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
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
1Scopus© Citations 22 18
Some of the metrics are blocked by your
consent settings
WNK3 is a Putative Chloride-sensing Kinase
(2011)
Pacheco Álvarez, Diana
;
Gamba, Gerardo
The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex. Copyright © 2011 S. Karger AG, Basel. © Cellular Physiology and Biochemistry
1 9Scopus© Citations 37
Some of the metrics are blocked by your
consent settings
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
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
Scopus© Citations 19 1 5

Pacheco Álvarez, Diana

Filters

Settings

Sort By

Results per page