Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains

BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes

A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane

BACKGROUND: ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix. METHODOLOGY/PRINCIPAL FINDINGS: Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. CONCLUSIONS: The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure

Characterization of human ClC-6, an intracellular chloride channel/transporter

ClC-6 is a member of the CLC protein family. This family comprises plasma membrane chloride channels and intracellular H+/Cl- antiporters which are conserved from bacteria to man. In human, the CLC family contains nine members: ClC-1 to -7, ClC-Ka and –Kb. Their physiological importance is underscored by several hereditary diseases that are linked to mutations in CLCs, such as myotonia congenita (ClC-1), epilepsy (ClC-2) or osteopetrosis (ClC-7). Whereas the CLCs that are expressed at the plasma membrane (endogenous or in heterologous expression systems) are well characterized, the ClC-6 channel/transporter, which is predominantly expressed intracellularly, remains one of the most enigmatic members of the family. In this study we unravelled some aspects of human ClC-6 (hClC-6). As classical patch clamping techniques are precluded for the functional study of this protein, we focused on the biochemistry and cellular biology of hClC-6. One of the fundamental steps of this thesis was the development and characterization of a polyclonal antibody against hClC-6. This antibody specifically recognized human and murine ClC-6 upon overexpression in Western blotting and immunocytochemistry. In addition, endogenous hClC-6 in a differentiated human neuroblastoma cell line, SH-SY5Y, could be detected in a specific manner by our antibody in immunocytochemistry. Experiments with detergent resistant membranes (DRM) suggested partitioning of hClC-6 in lipid rafts upon overexpression. Moreover, we identified a positively charged N-terminal sequence, KKGRR, which was involved in the DRM association of hClC-6. Mutation of the KKGRR sequence affected the colocalization of hClC-6 with hClC-7. Upon cotransfection, we observed that hClC-6 and hClC-7 could be spatially resolved in confocal laser scanning microscopy (CLSM), whereas the KKGRR-mutant hClC-6 and hClC-7 extensively colocalized. The molecular basis of this difference, however, needs to be further investigated. We also studied post-translational modifications of hClC-6. We found that hClC-6 could be N-glycosylated at three residues, N410, N422 and N432. These sites are located in a poorly conserved region of hClC-6 between the intramembrane helices K and M. Upon overexpression, only two of the three sites are N-glycosylated simultaneously, seemingly without any clear specific preference. We found that N-glycosylation was probably not involved in the initial sorting steps of ClC-6, but its contribution in the last sorting step from tubular endosomal network (TEN) to late endosomes remains to be elucidated. In addition, we studied putative phosphorylation events in a unique region in the cytoplasmic C-terminal tail and found in vitro phosphorylation by the closely related kinases PKA, PKB, PKC and PKD. In another series of experiments we studied the intracellular localization and sorting of hClC-6. In differentiated SH-SY5Y cells, endogenous hClC-6 localized to late endosomes/lysosomes, but upon overexpression the protein localized to the early/recycling endosomal compartment. We discovered that this discrepancy was caused by the overexpression, regardless of the cell type the protein was overexpressed in. We therefore suggest that hClC-6 is sorted from early endosomes to the TEN, which is a recently proposed model for sorting of proteins. In this scenario, hClC-6 is sorted from the TEN to late endosomes via the so-called lysosomal bypass pathway, and this last step is probably the rate limiting step that causes accumulation in TEN upon overexpression. We also investigated how ClC-6 is sorted to the endosomes. Surface biotinylation experiments demonstrated that hClC-6 was not expressed at the cell surface, or at least not at detectable levels. This experiment suggests that hClC-6 is sorted directly to the endosomes. Moreover, the identification of two dileucine motifs, reminiscent for TGN-to-endosomes sorting, confirmed this hypothesis and led to the finding that these motifs cooperated in the sorting out of the TGN. In conclusion, we propose that the sorting of ClC-6 to its endosomal compartment is a regulated multi-step process. We discuss several possible functions, but the role of ClC-6 remains quite enigmatic.status: publishe

Durable restoration of reinforced concrete grillage roof shell of Ensor Gallery in Ostend (B)

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Structureel gedrag van monumenten in Diestiaan ijzerzandsteen

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Modelling of damage accumulation in masonry subjected to a long term compressive load

A rheological model is used to describe the long-term behaviour of masonry. Special attention is paid to the evolution of damage and the description of the damage parameters, based on the continuum damage theory. The parameters of the model are derived from a test program, including three types of mortar compositions and three kinds of tests: monotonic compressive, short-term and long-term creep tests. Furthermore, a range of stress evolutions is simulated to evaluate the model and the results are compared with experimental test datastatus: publishe

Consolidation and strengthening of historical masonry: Modeling structural behaviour of grouted three-leaf masonry

Development of mineral grouts for consolidation and strengthening of historical masonry was discussed in Ref. [1]. The properties of the injection grout must counteract the elements that initiate the failure mechanism of multiple leaf historical masonry and lead to its collapse. This paper presents a macro-approach to model the structural behavior of three-leaf masonry. A global approach is used, based on the properties and the behavior of the external leafs, in combination with the properties and behavior of the central core of the threeleaf wall. Evaluation of the models is made by comparison with experimental data.status: publishe

Long-term behaviour of low quality sandstone masonry: assessment methodology and case study

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Standzekerheidsproblemen bij oude funderingssystemen, deel 1

edition: afl.status: publishe