Pacheco Álvarez, Diana
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Pacheco Álvarez, Diana
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Pacheco Álvarez, Diana
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dpacheco
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14827257500
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DVP-2845-2022

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The European and Japanese eel NaCl cotransporters β exhibit chloride currents and are resistant to thiazide type diuretics

2022 , Moreno, Erika , Plata, Consuelo , Vázquez, Norma , Oropeza-Viveros, Dulce María , Pacheco Álvarez, Diana , Rojas-Vega, Lorena , Olin-Sandoval, Viridiana , Gamba, Gerardo

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule, and the inhibition of its function with thiazides is widely used for the treatment of arterial hypertension. In mammals and teleosts, NCC is present as one ortholog that is mainly expressed in the kidney. One exception, however, is the eel, which has two genes encoding NCC. The eNCCα is located in the kidney and eNCCβ, which is present in the apical membrane of the rectum. Interestingly, the European eNCCβ functions as a Na+-Cl- cotransporter that is nevertheless resistant to thiazides and is not activated by low-chloride hypotonic stress. However, in the Japanese eel rectal sac, a thiazide-sensitive NaCl transport mechanism has been described. The protein sequences between eNCCβ and jNCCβ are 98% identical. Here, by site-directed mutagenesis, we transformed eNCCβ into jNCCβ. Our data showed that jNCCβ, similar to eNCCβ, is resistant to thiazides. In addition, both NCCβ proteins have high transport capacity with respect to their renal NCC orthologs and, in contrast to known NCCs, exhibit electrogenic properties that are reduced when residue I172 is substituted by A, G, or M. This is considered a key residue for the chloride ion-binding sites of NKCC and KCC. We conclude that NCCβ proteins are not sensitive to thiazides and have electrogenic properties dependent on Cl-, and site I172 is important for the function of NCCβ. ©American Journal of Physiology-Cell Physiology

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Aspectos estructurales, funcionales y patológicos del cotransportador de NaCl sensible a tiazidas

2014-11 , Moreno, Erika , Pacheco Álvarez, Diana , Ríos Argaiz, Eduardo

El cotransportador de Na-Cl sensible a tiazidas (NCC o CST) es la principal vía de reabsorción de sal en el túbulo distal de la nefrona y es el sitio de acción de los diuréticos de tipo tiazida que, por su utilidad en el manejo de la hipertensión arterial, se encuentran dentro de los medicamentos más recetados en el mundo. El NCC es una proteína de suma importancia para la fisiología renal, ya que permite mantener la homeostasis de sal y agua en el organismo. Cuando suceden mutaciones inactivantes en el gen que codifica para este cotransportador se produce una enfermedad conocida como síndrome de Gitelman, el cual es un trastorno autosómico recesivo caracterizado clínicamente por hipotensión arterial, alcalosis metabólica, hipocalemia e hipocalciuria, lo que resalta la importancia de este gen en la regulación de la presión arterial y el equilibrio hidroelectrolítico. En este trabajo hacemos una breve revisión de los conocimientos que se tienen acerca de este cotransportador, con especial énfasis en la biología molecular, propiedades fisiológicas y aspectos patológicos del NCC. ©Revista de Investigación Clínica

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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.

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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.

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Structure-function relationships in the sodium chloride cotransporter

2023 , Moreno, Erika , Pacheco Álvarez, Diana , Chávez-Canales, María , Elizalde, Stephanie , Leyva Ríos, Karla , Gamba, Gerardo

The thiazide sensitive Na+:Cl− cotransporter (NCC) is the principal via for salt reabsorption in the apical membrane of the distal convoluted tubule (DCT) in mammals and plays a fundamental role in managing blood pressure. The cotransporter is targeted by thiazide diuretics, a highly prescribed medication that is effective in treating arterial hypertension and edema. NCC was the first member of the electroneutral cation-coupled chloride cotransporter family to be identified at a molecular level. It was cloned from the urinary bladder of the Pseudopleuronectes americanus (winter flounder) 30 years ago. The structural topology, kinetic and pharmacology properties of NCC have been extensively studied, determining that the transmembrane domain (TM) coordinates ion and thiazide binding. Functional and mutational studies have discovered residues involved in the phosphorylation and glycosylation of NCC, particularly on the N-terminal domain, as well as the extracellular loop connected to TM7-8 (EL7-8). In the last decade, single-particle cryogenic electron microscopy (cryo-EM) has permitted the visualization of structures at high atomic resolution for six members of the SLC12 family (NCC, NKCC1, KCC1-KCC4). Cryo-EM insights of NCC confirm an inverted conformation of the TM1-5 and TM6-10 regions, a characteristic also found in the amino acid-polyamine-organocation (APC) superfamily, in which TM1 and TM6 clearly coordinate ion binding. The high-resolution structure also displays two glycosylation sites (N-406 and N-426) in EL7-8 that are essential for NCC expression and function. In this review, we briefly describe the studies related to the structure-function relationship of NCC, beginning with the first biochemical/functional studies up to the recent cryo-EM structure obtained, to acquire an overall view enriched with the structural and functional aspects of the cotransporter. Copyright © 2023 Moreno, Pacheco-Alvarez, Chávez-Canales, Elizalde, Leyva-Ríos and Gamba.

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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

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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.

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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

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