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 5

Characterization of intracellular trafficking defects of representative Cldn16 mutants.

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Characterization of intracellular trafficking defects of representative ...
Transfected MDCK cells expressing the indicated HA-tagged Cldn16 mutants were incubated at 20°C for 3 hours to allow transport out of the ER but prevent exit from the Golgi complex and TGN. Cells were then either processed for immunofluorescence staining (A and D) or transferred to 37°C for 1 hour (B and E) or 6 hours (C and F) in the presence of cycloheximide and ALLN to inhibit de novo protein synthesis and degradation, respectively. The cells were then immunostained with antibodies to HA to detect the tagged Cldn16 mutant (red) and either the Golgi marker GM130 (A, B, D, and E, green) or the lysosomal membrane protein CD63 (C and F, green). Shown are representative images of 2–3 independent experiments. For the remaining mutants, see Supplemental Figure 2.