Ding J et al. (2013), A trafficking-deficient mutant of KCC3 reveals ...

XB-ART-46934

PLoS One 2013 Jan 01;84:e61112. doi: 10.1371/journal.pone.0061112.

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A trafficking-deficient mutant of KCC3 reveals dominant-negative effects on K-Cl cotransport function.

Ding J , Ponce-Coria J , Delpire E .

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The K-Cl cotransporter (KCC) functions in maintaining chloride and volume homeostasis in a variety of cells. In the process of cloning the mouse KCC3 cDNA, we came across a cloning mutation (E289G) that rendered the cotransporter inactive in functional assays in Xenopus laevis oocytes. Through biochemical studies, we demonstrate that the mutant E289G cotransporter is glycosylation-deficient, does not move beyond the endoplasmic reticulum or the early Golgi, and thus fails to reach the plasma membrane. We establish through co-immunoprecipitation experiments that both wild-type and mutant KCC3 with KCC2 results in the formation of hetero-dimers. We further demonstrate that formation of these hetero-dimers prevents the proper trafficking of the cotransporter to the plasma membrane, resulting in a significant decrease in cotransporter function. This effect is due to interaction between the K-Cl cotransporter isoforms, as this was not observed when KCC3-E289G was co-expressed with NKCC1. Our studies also reveal that the glutamic acid residue is essential to K-Cl cotransporter function, as the corresponding mutation in KCC2 also leads to an absence of function. Interestingly, mutation of this conserved glutamic acid residue in the Na(+)-dependent cation-chloride cotransporters had no effect on NKCC1 function in isosmotic conditions, but diminished cotransporter activity under hypertonicity. Together, our data show that the glutamic acid residue (E289) is essential for proper trafficking and function of KCCs and that expression of a non-functional but full-length K-Cl cotransporter might results in dominant-negative effects on other K-Cl cotransporters.

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Species referenced: Xenopus laevis
Genes referenced: nlrp1 pdia2 slc12a2 slc12a5 slc12a6

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	Figure 2. Co-immunoprecipitation reveals interaction between KCC3 and KCC2.Xenopus laevis 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.
	Figure 3. Evidence for dominant-negative effect of KCC3 on KCC2 function.A) Xenopus laevis oocytes were injected with KCC2 cRNA in the presence or absence of wild-type KCC3 or KCC3-E289G cRNAs. Three days following injection, K+ influx was measured under isosmotic conditions where only KCC2 function is active, or under hyposmotic conditions where both KCC2 and KCC3 function are stimulated. Bars represent mean Â± S.E.M. (nâ=â20â25 oocytes). Flux is expressed in nmoles K+/oocyte/h. (*) P<0.01 (ANOVA) compared with KCC2 alone under isosmotic condition, (â) P<0.01 (ANOVA) compared with KCC2 alone under hyposmotic solution. Arrow indicates the anticipated flux mediated by the combined activity of KCC2 and KCC3. This is a representative experiment. Each condition (bar) was reproduced multiple times. B) K+ influx was measured under hyperosmotic conditions in oocytes injected with NKCC1 cRNA in the presence or absence of KCC3-E289G cRNA. Bars represent mean Â± S.E.M. (nâ=â20â25 oocytes). Flux is also expressed in nmoles K+/oocyte/h.
	Figure 4. Evidence for dominant-negative effect of KCC3 on KCC2 trafficking.Xenopus laevis 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.
	Figure 5. Evidence for dominant-negative effect of KCC3 on KCC2 trafficking and function.Xenopus laevis oocytes were injected with KCC3, KCC3-E289G, or both. A) Membrane fraction was isolated using a silica based cross-linking method and membrane proteins and whole oocyte lysates were subjected to Western blot analysis using rabbit polyclonal anti-KCC2 or anti-KCC3 antibodies. B) K+ influx was measured under hyposmotic conditions. C) K+ influx generated by KCC3-KCC3 and KCC3-KCC3-E289G concatamers. Bars represent mean Â± S.E.M. (nâ=â20â25 oocytes). Flux is expressed in nmoles K+/oocyte/h. *P<0.001 (ANOVA) compared with KCC3 controls. Experiment was performed 6 times with similar data.
	Figure 6. N-Glycolsylation deficiency of the KCC3-E289G mutant.A) Western blot analyses of KCC3-E289G mutant in HEK 293FT cells and Xenopus laevis oocytes compared to wild-type KCC3 in HEK 293FT cells, wild-type and mutant CHO cells, and Xenopus laevis 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.
	Figure 7. N-Glycolsylation deficiency of the KCC3-E289G mutant.A) Western blot analysis of wild-type KCC3 and KCC3-E289G mutant in HEK 293FT cells treated with tunicamycin (10 Âµg/ml for 18 h). B) Western blot analysis of wild-type KCC3 and KCC3-E289G mutant proteins isolated from Xenopus laevis oocytes and treated with PNGase (0.25U, 12 h at 37Â°C). The membranes were exposed to a rabbit polyclonal anti-KCC3 antibody. The experiment was repeated once with identical data.
	Figure 8. Evidence for KCC3-E289G localizing in the endoplasmic reticulum.HEK 293FT cells were transfected with wild-type KCC3 (AâF) or KCC3-E289G mutant (GâL). 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 Fluorâconjugated 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). Barâ=â5 Âµm.
	Figure 9. Sub-cellular localization of KCC3 and KCC3-E289G in HEK 293FT cells.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.
	Figure 10. Effect of E289G-like mutations in KCC2 and NKCC1.K+ influx was measured through unidirectional 86Rb tracer uptake in oocytes injected with water, wild-type KCC2 or KCC2-E201G or KCC2-E201D mutant cRNAs, and wild-type or NKCC1-E383G or NKCC1-E383D mutant cRNAs. Uptakes were measured under isosmotic (200 mOsM) and hypotonic (100 mOsM) solutions for KCC2 and under isotonic and hypertonic (265 mOsM) solutions for NKCC1. Bars represent meansÂ±SEM (nâ=â20â25 oocytes). Fluxes are expressed in nmoles K+/oocyte/h. (*) Denotes statistical significance with P<0.001 (ANOVA followed by Tukey-Kramer Multiple Comparisons Test). Experiment was done once.
	Figure 1. Absence of function of KCC3-E289G mutant cDNA.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 meansÂ±SEM (nâ€Š=â€Š25 oocytes).

References [+] :

Adragna, Hypertension in K-Cl cotransporter-3 knockout mice. 2004, Pubmed