Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport function of Claudin-16
P. Jaya Kausalya, Salah Amasheh, Dorothee Günzel, Henrik Wurps, Dominik Müller, Michael Fromm, Walter Hunziker
P. Jaya Kausalya, Salah Amasheh, Dorothee Günzel, Henrik Wurps, Dominik Müller, Michael Fromm, Walter Hunziker
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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 6

ER-retained Cldn16 mutants are subject to proteasomal degradation.

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ER-retained Cldn16 mutants are subject to proteasomal degradation. (A–F)...
(A–F) Colocalization of ER-retained Cldn16 mutants with ubiquitin was increased in the presence of a proteasome inhibitor. Transfected MDCK cells expressing HA-tagged Cldn16 or the indicated mutants were incubated for 10 hours in the absence (A–C) or presence (D–F) of the proteasome inhibitor ALLN. Cells were then immunostained with antibodies to detect the HA-tagged Cldn16 mutant (red) or ubiquitin (Ub, green). Colocalization was apparent in the merged images (yellow). Shown are representative images of 2–3 independent experiments. For the remaining mutants, see Supplemental Figure 3. (G) ER-retained Cldn16 mutants were stabilized by proteasome inhibitors. HEK-293T cells transiently expressing the indicated HA-tagged Cldn16 mutants were incubated in the absence (–) or presence (+) of the proteasome inhibitor ALLN. Cells were then lysed, and Cldn16 was detected by SDS-PAGE and Western blot analysis. Blotting for actin was used as a loading control (data not shown). (H) Turnover of ER-retained Cldn16 mutants was blocked by proteasome inhibitors. HEK-293T cells transiently expressing HA-tagged Cldn16 or the ER-retained HA-tagged G92V were cultured in cycloheximide to block de novo protein synthesis for the time periods indicated, either in the absence or presence of the proteasome inhibitor ALLN. Cells were then lysed, and Cldn16 detected by SDS-PAGE and Western blot analysis. Percent expression relative to that at 0 hours is shown below each lane. Blotting for actin was used as a loading control. One representative blot and quantification of 3 independent experiments is shown.