Regulación del cotransportador Na+/K+/2Cl- NKCC2 por la concentración de cloruro intracelular / tesis que para obtener el grado de Doctor en Ciencias Bioquímicas, presenta José Ponce Coria ; asesor Gerardo Gamba Ayala

. xvi, 180 páginas : ilustraciones. Doctorado en Ciencias Bioquímicas UNAM, Facultad de Química, 201

Evidence for KCC3-E289G localizing in the endoplasmic reticulum.

<p>HEK 293FT cells were transfected with wild-type KCC3 (A–F) or KCC3-E289G mutant (G–L). Two days post- transfection, the cells were fixed with paraformaldehyde, treated with saponin, and exposed to rabbit polyclonal anti-KCC3 and mouse monoclonal anti-PDI antibodies followed by cy3-conjugated anti-rabbit and Alexa Fluor–conjugated goat anti-mouse antibodies. Focal plane images of KCC3 signal (A, D, J, G), ER marker signal (B, E, H, K), and combined signals (C, F, I, L). Bar = 5 µm.</p

N-Glycolsylation deficiency of the KCC3-E289G mutant.

<p>A) Western blot analysis of wild-type KCC3 and KCC3-E289G mutant in HEK 293FT cells treated with tunicamycin (10 µg/ml for 18 h). B) Western blot analysis of wild-type KCC3 and KCC3-E289G mutant proteins isolated from <i>Xenopus laevis</i> oocytes and treated with PNGase (0.25U, 12 h at 37°C). The membranes were exposed to a rabbit polyclonal anti-KCC3 antibody. The experiment was repeated once with identical data.</p

Sub-cellular localization of KCC3 and KCC3-E289G in HEK 293FT cells.

<p>HEK 293FT cells were transfected with wild-type KCC3 or KCC3-E289G mutant. Two days post-transfection, the cells were treated with digitonin to extract proteins from cholesterol-rich (plasma) membranes (membrane/cytosol fraction), followed by NP40 treatment to isolate proteins from ER/Golgi fraction, followed by deoxycholate+SDS detergents to isolate proteins from nuclear fraction. Western blots were probed with rabbit polyclonal anti-KCC3 and mouse monoclonal anti-PDI and anti-GAPDH antibodies. Experiment was reproduced at least 5 times with similar data.</p

Evidence for dominant-negative effect of KCC3 on KCC2 trafficking.

<p><i>Xenopus laevis</i> oocytes were injected with KCC2 in the presence or absence of wild-type KCC3 or KCC3-E289G cRNAs and membrane fractions were isolated using a silica cross-linking method. Membrane proteins and whole oocyte lysates were subjected to Western blot analysis using rabbit polyclonal anti-KCC2 or anti-KCC3 antibodies. Experiment was reproduced 3 times.</p

Co-immunoprecipitation reveals interaction between KCC3 and KCC2.

<p><i>Xenopus laevis</i> oocytes were injected with KCC2 cRNA in the presence or absence of wild-type KCC3 or KCC3-E289G cRNAs. KCC2 or KCC3 were then immunoprecipitated and the complex was analyzed by Western blot analysis using rabbit polyclonal anti-KCC2 or anti-KCC3 antibodies. Immunodetection of KCC2, KCC3 and IgG are indicated on the right of the panels. Note that both KCC2 (panel A) and KCC3 (panel B) when immunoprecipitated are observed as 2 bands: unglycosylated and glycosylated forms. Experiment was repeated once and yielded similar data.</p

Absence of function of KCC3-E289G mutant cDNA.

<p>A) Conservation of glutamic acid residue (E289 in mouse KCC3) within mouse SLC12A cotransporters. The residue, highlighted by an arrowhead in cartoon and sequence alignment, is localized at the end or right downstream of transmembrane domain 3 (TM3). B) K⁺ influx was measured through unidirectional ⁸⁶Rb tracer uptake under isosmotic and hyposmotic conditions, in oocytes injected with water, KCC3-E289G mutant cRNA, and wild-type KCC3 cRNA. Bars represent means±SEM (n = 25 oocytes).</p

N-Glycolsylation deficiency of the KCC3-E289G mutant.

<p>A) Western blot analyses of KCC3-E289G mutant in HEK 293FT cells and <i>Xenopus laevis</i> oocytes compared to wild-type KCC3 in HEK 293FT cells, wild-type and mutant CHO cells, and <i>Xenopus laevis</i> oocytes, using rabbit polyclonal anti-KCC3 antibody. CHO-Lec1 cells have mutation in N-acetylglucosaminyl transferase, whereas CHO-Lec8 and CHO-Lec2 have deficient galactose and sialic acid transporters, respectively. Two independent experiments are shown. Experiment was performed 4 times. B) Scheme represents the main steps in N-linked oligosaccharide biosynthetic pathway. First, core Glc-Nac-Glc-Nac-Man with branched mannose residues are added to the Asparagines in the ER. In the Golgi, mannose molecules are replaced by acethylglucosamyl groups, followed by the addition of galactose and sialic acid groups. These steps require the availability of galactose and sialic acid in the cells, which is prevented in the mutant CHO cells by elimination of specific transporters.</p

WNK Kinases, Renal Ion Transport and Hypertension

Two members of a recently discovered family of protein kinases are the cause of an inherited disease known as pseudohypoaldosteronism type II (PHAII). These patients exhibit arterial hypertension together with hyperkalemia and metabolic acidosis. This is a mirror image of Gitelman disease that is due to inactivating mutations of the SLC12A3 gene that encodes the thiazide-sensitive Na+: Cl− cotransporter. The uncovered genes causing PHAII encode for serine/threonine kinases known as WNK1 and WNK4. Physiological and biochemical studies have revealed that WNK1 and WNK4 modulate the activity of several transport pathways of the aldosterone-sensitive distal nephron, thus increasing our understanding of how diverse renal ion transport proteins are coordinated to regulate normal blood pressure levels. Observations discussed in the present work place WNK1 and WNK4 as genes involved in the genesis of essential hypertension and as potential targets for the development of antihypertensive drugs