Phosphorylated claudin-16 interacts with Trpv5 and regulates transcellular calcium transport in the kidney

Abstract

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) was previously considered to be a paracellular channelopathy caused by mutations in the claudin-16 and claudin-19 genes. Here, we provide evidence that a missense FHHNC mutation c.908C>G (p.T303R) in the claudin-16 gene interferes with the phosphorylation in the claudin-16 protein. The claudin-16 protein carrying phosphorylation at residue T303 is localized in the distal convoluted tubule (DCT) but not in the thick ascending limb (TAL) of the mouse kidney. The phosphomimetic claudin-16 protein carrying the T303E mutation but not the wildtype claudin-16 or the T303R mutant protein increases the Trpv5 channel conductance and membrane abundance in human kidney cells. Phosphorylated claudin-16 and Trpv5 are colocalized in the luminal membrane of the mouse DCT tubule; phosphomimetic claudin-16 and Trpv5 interact in the yeast and mammalian cell membranes. Knockdown of claudin-16 gene expression in transgenic mouse kidney delocalizes Trpv5 from the luminal membrane in the DCT. Unlike wildtype claudin-16, phosphomimetic claudin-16 is delocalized from the tight junction but relocated to the apical membrane in renal epithelial cells because of diminished binding affinity to ZO-1. High-Ca2+ diet reduces the phosphorylation of claudin-16 protein at T303 in the DCT of mouse kidney via the PTH signaling cascade. Knockout of the PTH receptor, PTH1R, from the mouse kidney abrogates the claudin-16 phosphorylation at T303. Together, these results suggest a pathogenic mechanism for FHHNC involving transcellular Ca2+ pathway in the DCT and identify a molecular component in renal Ca2+ homeostasis under direct regulation of PTH.

Structure, Function, and Regulation of the Blood-Brain Barrier Tight Junction in Central Nervous System Disorders

“…Mass spectrometry (MS) data have revealed multiple phosphorylation sites in the C-terminal region of many claudins ( Hornbeck et al, 2015 ; Van Itallie and Anderson, 2018 ). Although the regulatory role of claudin phosphorylation has not yet been directly studied at the brain microvascular endothelium, studies in other epithelial and endothelial tissues have shown roles in modulation of protein-protein interactions ( Nomme et al, 2015 ), intracellular trafficking ( Van Itallie et al, 2012 ; Twiss et al, 2013 ), and protein turnover mechanisms ( Mandel et al, 2012 ; Hou et al, 2019 ). For example, Rho kinase (RhoK) has been demonstrated by Yamamoto et al (2008) to phosphorylate claudin-5 at tyrosine 207 (T207) in mouse and human brain tissues.…”

Pathophysiological aspects of the thick ascending limb and novel genetic defects: HELIX syndrome and transient antenatal Bartter syndrome

“…Other types of interactions, such as those between different nephron segments, seem to play roles in the physiopathologies of known diseases of the TAL as shown by the recent work of Hou et al [71]. These authors showed that phosphorylated claudin-16 is present in luminal membrane of the distal convoluted tubule, where it interacts with the calcium channel Trpv5 to increase the transcellular calcium reabsorption.…”

Claudin-10a Deficiency Shifts Proximal Tubular Cl- Permeability to Cation Selectivity via Claudin-2 Redistribution

“…Enzymatic tubule preparation for molecular biology was performed as follows: thin transversal slices were digested in incubation solution (in mM: 140 NaCl, 0.4 KH 2 PO 4 , 1.6 K 2 HPO 4 , 1 MgSO 4 , 10 sodium acetate, 1 α-ketoglutarate, 1.3 calcium gluconate, 5 glycine, pH 7.4, containing 48 µg/ml trypsin inhibitor and 25 µg/ml DNase I) with 2 mg/ml collagenase II (PAN-Biotech, Aidenbach, Germany) at 37°C and 850 rpm in a thermoshaker for 15–25 minutes. After vigorous washing in sorting solution (incubation solution supplemented with 0.5 µg/ml BSA), tubules were transferred to stereomicroscopes (Leica, Wetzlar, Germany) and sorted at 4°C according to morphologic criteria 27 – 29 . For RNA preparation 10 PCTs and approximately 20 distal convoluted tubules (DCTs) per kidney were pooled, snap frozen, and stored at −80°C until further processing for RNA sequencing (RNA-Seq).…”