Biomedical subjects

Norma Vázquez

Publications and source records attributed to Norma Vázquez.

12 recordsLinked to original sources

PubMed2006

WNK4 kinase is a negative regulator of K+-Cl- cotransporters.

WNK kinases [with no lysine (K) kinase] are emerging as regulators of several membrane transport proteins in which WNKs act as molecular switches that coordinate the activity of several players. Members of the cation-coupled chloride cotransporters family (solute carrier family number 12) are one of the main targets. WNK3 activates the Na(+)-driven cotransporters NCC, NKCC1, and NKCC2 and inhibits the K(+)-driven cotransporters KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine-rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 on the K(+)-Cl(-) cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K(+)-Cl(-) cotransporters KCC1, KCC3, and KCC4 under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. In contrast, three different WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC2 and KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK, was prevented by the presence of protein phosphatase inhibitors, and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K(+)-Cl(-) cotransporters expressed in the kidney.

Animals↗

PubMed2006

Affinity-defining domains in the Na-Cl cotransporter: a different location for Cl- and thiazide binding.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na+, Cl-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different.

Amino Acid Sequence↗

PubMed2006

WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway.

SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular Cl- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl- influx and efflux branches of the SLC12A cotransporter family.

Animals↗

PubMed2005

Activity of the renal Na+-K+-2Cl- cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl- transport.

The renal-specific Na(+)-K(+)-2Cl(-) cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na(+)-Cl(-) cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by (86)Rb(+) influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced approximately 20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl(-) affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

Animals↗

PubMed2005

NH2-terminal heterogeneity in the KCC3 K+-Cl- cotransporter.

The SLC12A6 gene encoding the K(+)-Cl(-) cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH(2)-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5' of two distinct first coding exons. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both the mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of NH(2)-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in X. laevis oocytes reveals apparent K(m)s for Rb(+) and Cl(-) of 10.7 +/- 2.5 and 7.3 +/- 1.2 mM, respectively, with an anion selectivity of Br(-) > Cl(-) > PO(4) = I(-) = SCN(-) = gluconate. All five NH(2)-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S proteins. In summary, there is significant NH(2)-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the kidney. Basolateral swelling-activated K(+)-Cl(-) cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.

Amino Acid Sequence↗

PubMed2004

Ion and diuretic specificity of chimeric proteins between apical Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters.

The mammalian kidney bumetanide-sensitive Na(+)-K(+)-2Cl(-) and thiazide-sensitive Na(+)-Cl(-) cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters to design and study chimeric proteins in which the NH(2)-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na(+)-K(+)-2Cl(-) cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH(2)-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na(+)-Cl(-) cotransporter.

Animals↗

PubMed2004

Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease.

Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties.

Alkalosis↗

PubMed2004

A single nucleotide polymorphism alters the activity of the renal Na+:Cl- cotransporter and reveals a role for transmembrane segment 4 in chloride and thiazide affinity.

The thiazide-sensitive Na+:Cl- cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physiological studies on pressure natriuresis and by its involvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na+:Cl- cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 is a highly conserved residue, we studied the functional properties of this polymorphism by using in vitro mutagenesis and the heterologous expression system in Xenopus laevis oocytes. G264A resulted in a significant and reproducible reduction ( approximately 50%) in (22)Na+ uptake when compared with the wild type cotransporter. The affinity for extracellular Cl- and for thiazide diuretics was increased in G264A. Western blot analysis showed similar immunoreactive bands between the wild type and the G264A cotransporters, and confocal images of oocytes injected with enhanced green fluorescent protein-tagged wild type and G264A cotransporter showed no differences in the protein surface expression level. These observations suggest that the G264A polymorphism is associated with reduction in the substrate translocation rate of the cotransporter, due to a decrease in the intrinsic activity. Our study also reveals a role of the transmembrane segment 4 in defining the affinity for extracellular Cl- and thiazide diuretics.

Adult↗

PubMed2003

cAMP-dependent activation of the renal-specific Na+-K+-2Cl- cotransporter is mediated by regulation of cotransporter trafficking.

The murine apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter gene (mBSC1) exhibits two spliced isoform products that differ at the COOH-terminal domain. A long COOH-terminal isoform (L-mBSC1) encodes the Na(+)-K(+)-2Cl(-) cotransporter, and a short isoform (S-mBSC1) exerts a dominant-negative effect on L-mBSC1 cotransporter activity that is abrogated by cAMP. However, the mechanism of this dominant-negative effect was not clear. In this study, we used confocal microscopic analysis of an enhanced green fluorescent protein (EGFP) fusion construct (L-mBSC1-EGFP) expressed to characterize the surface expression of the L-BSC1 isoform in Xenopus laevis oocytes. Functional expression was also assessed in L-mBSC1-injected oocytes by measuring the bumetanide-sensitive (86)Rb(+) uptake. Oocytes injected with L-mBSC1-EGFP cRNA developed a distinct plasma membrane-associated fluorescence that colocalized with the fluorescent membrane dye FM 4-64. The fluorescence intensity in L-mBSC1-EGFP oocytes did not change after cAMP was added to the extracellular medium. In contrast, L-mBSC1-EGFP fluorescence intensity was reduced in a dose-dependent manner, with coexpression of S-mBSC1. The inhibitory effect of S-mBSC1 was abrogated by cAMP. Finally, the exocytosis inhibitor colchicine blocked the effect of cAMP on the L-mBSC1-EGFP/S-mBSC1-coinjected oocytes. All changes in L-mBSC1 surface expression correlated with modification of bumetanide-sensitive (86)Rb(+) uptake. Our data suggest that the dominant-negative effect of S-mBSC1 on L-mBSC1 transport function is due to the effects of the cotransporter on trafficking.

1-Methyl-3-isobutylxanthine↗

PubMed2003

N-Glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide-sensitive Na(+):Cl(-) cotransporter.

The rat thiazide-sensitive Na-Cl cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N-linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the high-mannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated cotransporter. Functional expression of cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive (22)Na(+) uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGFP)-tagged cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl(-) affinity. Thus, we have demonstrated that rNCC is N-glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site.

Animals↗

PubMed2002

Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter.

The expression level of the neuronal-specific K-Cl cotransporter KCC2 (SLC12A5) is a major determinant of whether neurons will respond to GABA with a depolarizing, excitatory response or a hyperpolarizing, inhibitory response. In view of the potential role in human neuronal excitability we have characterized the hKCC2 cDNA and gene. The 5.9 kb hKCC2 transcript is specific to brain, and is induced during in vitro differentiation of NT2 teratocarcinoma cells into neuronal NT2-N cells. The 24-exon SLC12A5 gene is on human chromosome 20q13, and contains a polymorphic dinucleotide repeat within intron 1 near a potential binding site for neuron-restrictive silencing factor. Expression of hKCC2 cRNA in Xenopus laevis oocytes results in significant Cl(-)-dependent (86)Rb(+) uptake under isotonic conditions; cell swelling under hypotonic conditions causes a 20-fold activation, which is blocked by the protein phosphatase inhibitor calyculin-A. In contrast, oocytes expressing mouse KCC4 do not mediate isotonic K-Cl cotransport but express much higher absolute transport activity than KCC2 oocytes under hypotonic conditions. Initial and steady state kinetics of hKCC2-injected oocytes were performed in both isotonic and hypotonic conditions, revealing K(m)s for K(+) and Cl(-) of 9.3+/-1.8 mM and 6.8+/-0.9 mM, respectively; both affinities are significantly higher than KCC1 and KCC4. The K(m) for Cl(-) is close to the intracellular Cl(-) activity of mature neurons, as befits a neuronal efflux mechanism.

Amino Acid Sequence↗

PubMed2002

Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter.

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161-F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na(+) than for Cl(-), with apparent Michaelis-Menten constant (K(m)) values of 58.2 +/- 7.1 and 22.1 +/- 4.2 mM, respectively. These K(m) values are significantly higher than those observed in rTSC. The Na(+) and Cl(-) affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10(-9) to 10(-7) M) resulted in activation of the cotransporter, whereas higher concentrations (10(-6) to 10(-4) M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na(+) or the Cl(-) concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg(2+), with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.

Animals↗