Transit Defect of Potassium-Chloride Co-transporter 3 Is a Major Pathogenic Mechanism in Hereditary Motor and Sensory Neuropathy with Agenesis of the Corpus Callosum

Centre of Excellence in Neuromics, University of Montreal, Centre Hospitalier de l'Université de Montréal-Research Center, Montreal, Quebec H2L 4M1, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2011; 286(32):28456-65. DOI: 10.1074/jbc.M111.226894
Source: PubMed


Missense and protein-truncating mutations of the human potassium-chloride co-transporter 3 gene (KCC3) cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe neurodegenerative
disease characterized by axonal dysfunction and neurodevelopmental defects. We previously reported that KCC3-truncating mutations
disrupt brain-type creatine kinase-dependent activation of the co-transporter through the loss of its last 140 amino acids.
Here, we report a novel and more distal HMSN/ACC-truncating mutation (3402C→T; R1134X) that eliminates only the last 17 residues
of the protein. This small truncation disrupts the interaction with brain-type creatine kinase in mammalian cells but also
affects plasma membrane localization of the mutant transporter. Although it is not truncated, the previously reported HMSN/ACC-causing
619C→T (R207C) missense mutation also leads to KCC3 loss of function in Xenopus oocyte flux assay. Immunodetection in Xenopus oocytes and in mammalian cultured cells revealed a decreased amount of R207C at the plasma membrane, with significant retention
of the mutant proteins in the endoplasmic reticulum. In mammalian cells, curcumin partially corrected these mutant protein
mislocalizations, with more protein reaching the plasma membrane. These findings suggest that mis-trafficking of mutant protein
is an important pathophysiological feature of HMSN/ACC causative KCC3 mutations.

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Available from: Masoud Shekarabi, Nov 09, 2015
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    • "C-terminal domain truncation of KCC3 and its defective transit to the plasma membrane are the major pathogenic mechanisms in HMSN/ACC that lead to the inactivation of the cotransporter, which fails to respond to swelling [1], [4]–[6]. We have determined that KCC3 truncation disrupts functional protein-protein interactions, such as KCC3 interaction with the brain-type creatine kinase (CK-B) [5], [6]. However, a co-morbid effect of KCC3 loss-of-function is the aberrant pathfinding of callosal axons, which migrate along the brain midline to form Probst's bundles instead of bridging the two brain hemispheres [3]. "
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    ABSTRACT: Loss-of-function of the potassium-chloride cotransporter 3 (KCC3) causes hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), a severe neurodegenerative disease associated with defective midline crossing of commissural axons in the brain. Conversely, KCC3 over-expression in breast, ovarian and cervical cancer is associated with enhanced tumor cell malignancy and invasiveness. We identified a highly conserved proline-rich sequence within the C-terminus of the cotransporter which when mutated leads to loss of the KCC3-dependent regulatory volume decrease (RVD) response in Xenopus Laevis oocytes. Using SH3 domain arrays, we found that this poly-proline motif is a binding site for SH3-domain containing proteins in vitro. This approach identified the guanine nucleotide exchange factor (GEF) Vav2 as a candidate partner for KCC3. KCC3/Vav2 physical interaction was confirmed using GST-pull down assays and immuno-based experiments. In cultured cervical cancer cells, KCC3 co-localized with the active form of Vav2 in swelling-induced actin-rich protruding sites and within lamellipodia of spreading and migrating cells. These data provide evidence of a molecular and functional link between the potassium-chloride co-transporters and the Rho GTPase-dependent actin remodeling machinery in RVD, cell spreading and cell protrusion dynamics, thus providing new insights into KCC3's involvement in cancer cell malignancy and in corpus callosum agenesis in HMSN/ACC.
    Full-text · Article · May 2013 · PLoS ONE
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    • "Injection of KCC3-T813X, the prevalent mutation observed in the French-Canadian population, in Xenopus laevis oocytes demonstrated expression of a glycosylated protein of a smaller molecular size at or near the oocyte plasma membrane similar to wild-type KCC3 [16]. In contrast, a novel and more distal HMSN/ACC truncating mutant (KCC3-R1134X) failed to traffic properly to the plasma membrane [24]. "
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    ABSTRACT: 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.
    Full-text · Article · Apr 2013 · PLoS ONE
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    ABSTRACT: The homologous genes encoding the electroneutral solute carrier family 12A (SLC12A) were identified more than 20 years ago, however, over the last few years, it has become clear that each of the genes within this family potentially encode for more than one cation-chloride cotransporter (CCC). Even more surprising, despite more than 30 years of functional studies and a wealth of knowledge on the activators, inhibitors, ion affinities, and kinetics of these cotransporters, we still cannot sufficiently explain why some cells express only one CCC isoform, while others express two, three, or more CCC isoforms. In 2009, Drs. Alvarez-Leefmans and Di Fulvio published an extensive in silico molecular analysis of the potential splice variants of the Na(+)-dependent cation-chloride cotransporters. In this review, we will look at the exceptionally large variety of potential splice variants within the Na(+)-independent cation-chloride cotransporter (SLC12A4-SLC12A7) genes, their initial tissue identification, and their physiological relevance. © 2014 S. Karger AG, Basel.
    Full-text · Article · Dec 2013 · Cellular Physiology and Biochemistry
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