Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport function of Claudin-16
P. Jaya Kausalya, … , Michael Fromm, Walter Hunziker
P. Jaya Kausalya, … , Michael Fromm, Walter Hunziker
Published April 3, 2006
Citation Information: J Clin Invest. 2006;116(4):878-891. https://doi.org/10.1172/JCI26323.
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Research Article Nephrology

Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport function of Claudin-16

Abstract

Claudin-16 (Cldn16) is selectively expressed at tight junctions (TJs) of renal epithelial cells of the thick ascending limb of Henle’s loop, where it plays a central role in the reabsorption of divalent cations. Over 20 different mutations in the CLDN16 gene have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a disease of excessive renal Mg2+ and Ca2+ excretion. Here we show that disease-causing mutations can lead to the intracellular retention of Cldn16 or affect its capacity to facilitate paracellular Mg2+ transport. Nine of the 21 Cldn16 mutants we characterized were retained in the endoplasmic reticulum, where they underwent proteasomal degradation. Three mutants accumulated in the Golgi complex. Two mutants were efficiently delivered to lysosomes, one via clathrin-mediated endocytosis following transport to the cell surface and the other without appearing on the plasma membrane. The remaining 7 mutants localized to TJs, and 4 were found to be defective in paracellular Mg2+ transport. We demonstrate that pharmacological chaperones rescued surface expression of several retained Cldn16 mutants. We conclude that FHHNC can result from mutations in Cldn16 that affect intracellular trafficking or paracellular Mg2+ permeability. Knowledge of the molecular defects associated with disease-causing Cldn16 mutations may open new venues for therapeutic intervention.

Authors

P. Jaya Kausalya, Salah Amasheh, Dorothee Günzel, Henrik Wurps, Dominik Müller, Michael Fromm, Walter Hunziker

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Figure 2

Clathrin-mediated endocytosis of Cldn16.

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Clathrin-mediated endocytosis of Cldn16. (A–C) Characterization of an an...
(A–C) Characterization of an antibody to the first extracellular loop of Cldn16 (α-loop) and endogenous Cldn16 expression in MDCK cells. MDCK cells were incubated for 1 hour at 37°C in the presence of anti-loop antibody alone (A), together with antigenic peptide (B), or with preimmune serum (C). Cells were then washed, fixed, and permeabilized, and the anti-loop antibody was detected by immunofluorescence microscopy using a labeled secondary antibody. (D–F) Detection of HA-tagged Cldn16 expressed in MDCK cells. MDCK cells expressing N-terminally HA epitope–tagged Cldn16 were incubated for 1 hour at 37°C with anti-loop antibody. α-HA (D) and the anti-loop antibody (E) were then detected in permeabilized cells. (F) Merging the HA and anti-loop antibody staining shows extensive colocalization at the cell surface and in a few intracellular vesicles. (G–I) Detection of anti-loop antibody internalized via HA-tagged Cldn16 in early endosomes of transfected HeLa cells. Anti-loop antibody (G) and EEA1 (H) were visualized by immunofluorescence microscopy, and the 2 images were merged (I). (J–L) Clathrin-mediated internalization of Cldn16. HeLa cells expressing HA-tagged Cldn16 were incubated for 1 hour at 37°C with anti-loop antibody under either hypertonic (J) or cytosol acidified (K) conditions to disrupt clathrin function or in the presence of cholesterol oxidase (L) to disrupt caveolae. Cldn16 was then visualized by immunofluorescence microscopy. In L, cells were stained with antibodies to EEA1, and the merged image is shown. Shown are representative images of 2–3 independent experiments.