Methods to analyze subcellular localization and intracellular trafficking of Claudin-16

The integral tight junction protein Claudin-16 (Cldn16) is predominantly expressed in renal epithelial cells of the thick ascending limb of Henle's loop where, together with claudin-19, it forms a cation-selective pore that allows influx of Na+ from the interstitial fluid into the lumen of the kidney tubule. This leads to an electrochemical gradient that drives the reabsorbtion of Mg2+ and Ca2+ ions from the renal filtrate. Mutations in the Cldn16 gene have been identified in patients suffering from familial hypomagnesemia with hypercalciuria and nephrocalcinosis, with excessive renal wastage of Mg2+ and Ca2+ being a hallmark of this condition. Studies into the mechanism by which mutations impair Cldn16 function have shown that although several mutations affect paracellular ion transport, many interfere with intracellular trafficking of Cldn16, ultimately compromising its localization to TJs. Here, we describe the experimental approaches that can be used to monitor intracellular localization and trafficking of Cldn16. These methods can easily be adapted to study other claudins, provided suitable antibodies are available.