Leyva Ríos, Karla
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Leyva Ríos, Karla
Official Name
Leyva Ríos, Karla
Alternative Name
kleyva
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Scopus Author ID
57191611929
Researcher ID
FIP-7416-2022

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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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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.

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