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.

Biology of claudins

“…However, Ca 2ϩ transport was strikingly asymmetrical (exclusively apical-to-basal), inhibited by Mg 2ϩ , and only slightly increased above control levels, raising some doubt as to whether it was truly paracellular or specific. By contrast, Kausalya et al (57) found that claudin-16 modestly increased Mg 2ϩ permeability (P Mg ) without changing P Na , and Hou et al (44) found a marked increase in P Na with only a minor increase in P Mg . Similarly, while we have found that claudin-19, when expressed in MDCKII cells, decreases permeability to Na ϩ , Ca 2ϩ , and Mg 2ϩ (6), Hou et al (45) found that claudin-19 transfection into LLC-PK 1 cells reduced only Cl Ϫ permeability (P Cl ).…”

“…At least 30 different claudin-16 mutations have now been reported in families with FHHNC (13, 56, 60, 65, 66, 76 -78, 88, 91, 94, 95, 100, 110, 111). Several are predicted to prematurely truncate the protein, and many have been shown to cause defects in trafficking of the protein to the plasma membrane (44,57,60). These findings suggested that claudin-16 might function as a divalent cation-selective paracellular pore and that FHHNC could be due to loss-of-function mutations that would be expected to abolish paracellular P Ca and P Mg in the TALH.…”

Magnesium in the Central Nervous System

“…The gene responsible for this disease was identified by Lifton and collaborators in 1999 and named Paracellin--1 (PCLN--1) . More than 20 mutations affecting paracellin--1 trafficking or permeability have been identified up--to--date (Kausalya et al, 2006). PCLN--1 encodes for paracellin--1 (PCLN--1), also termed claudin--16.…”

“…SSRIs (Eller et al, 2008;O'Brien et al, 2007). Anti--inflammatory agents appear to have anti--depressant effects, as in the case of TNFα--antagonists (Uguz et al, 2009), cyclooxygenase inhibition (Muller et al, 2006) and omega--3 fatty acids .…”

“…The HPA axis has long been recognized as a potential target for antidepressants (Flores et al, 2006;Holsboer, 2000;Jahn et al, 2004). More recently, anti--inflammatory mechanisms like COX--2 inhibitors (Muller et al, 2006) and TNFα--antagonists (Uguz et al, 2009) have been demonstrated. In the following text we report behavioural effects by Mg in animal models as well as human clinical data.…”