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.

Keywords: PTH; Trpv5; calcium; claudin; tight junction.

Fig. 1.

Phosphorylated claudin-16 protein localization in the kidney. Dual immunofluorescent staining of phosphorylated claudin-16 protein (CLDN16-P) with a TAL marker (THP), with a DCT1 marker (parvalbumin), with a DCT2 marker (calbindin-D28K), and with a CNT/CD marker (AQP2). (Scale bar: 20 μm.)

Fig. 2.

Effects of claudin-16 phosphorylation on membrane Ca²⁺ permeability in the presence or absence of Trpv5. Shown in the statistical graph are assays of [45]Ca²⁺ influx rates in cells expressing Trpv5 with various claudin-16 proteins, including wildtype (WT) and dephosphorylated (T303R) and phosphomimetic (T303E) claudin-16 (**P < 0.01; n = 5 transfections). n.s., not significant.

Fig. 3.

Claudin-16 mutant T303E increases Trpv5 current density. (A) The steady-state current-voltage (I-V) curve revealed a characteristic inwardly rectifying Trpv5 currents in HEK293 cells transfected with Trpv5 plus empty vector versus claudin-16 WT, T303E, or T303R mutant (n ≥ 5 recordings for each group). Trpv5 current density (current normalized to cell plasma membrane area, pA/pF; mean ± SEM) was evoked by test pulses from −150 to +100 mV, with +25-mV increments, for 200 ms. (B) In HEK293 cells coexpressing Trpv5 with claudin-16 variants, T303E but not WT or T303R claudin-16 increased TRPV5 current density compared with control (Trpv5+empty vector; P < 0.0001; n ≥ 5 recordings). Representative current traces are shown below each study group. n.s., not significant.

Fig. 4.

Claudin-16 mutant T303E increases Trpv5 membrane abundance levels. Cell-surface biotinylation assays of Trpv5 abundance levels in cells expressing Trpv5 with claudin-16 variant proteins, including wildtype (WT) and dephosphorylated (T303R) and phosphomimetic (T303E) claudin-16. Trpv5 proteins migrate as 2 separate bands on PAGE gel: 82-kDa core protein (white arrowhead) and 92-kDa glycosylated protein (black arrowhead; *P < 0.05; n = 3 transfections versus empty vector).

Fig. 5.

Trpv5 protein abundance and localization in claudin-16 KD mouse kidney. (A) Luminal membranes from mouse distal tubular cells were extracted, pooled, and assayed for Trpv5 protein abundance levels. Trpv5 proteins migrate as 2 separate bands on PAGE gel: 82-kDa core protein (white arrowhead) and 92-kDa glycosylated protein (black arrowhead). NCC proteins were used as loading control. (B) Whole-kidney Trpv5 mRNA levels were measured with real-time PCR (n.s., not significant; n = 5 animals). (C) Dual immunofluorescent staining of Trpv5 with the DCT2 marker (calbindin-D28K) on the kidney sections from WT and claudin-16 KD mice. The rabbit anti-Trpv5 from Alpha Diagnostics was used in this experiment. (Scale bar: 20 μm.)

Fig. 6.

Protein interaction of claudin-16 with Trpv5. (A) Y2H assays showing interaction of Trpv5 with CLDN16-WT, CLDN16-T303E, and CLDN16-T303R. Shown are plates with selective medium lacking leucine and tryptophan (−LW), indicating the transforming of both bait and prey vectors; with SD-LWH, indicating the expression of reporter gene HIS3; and β-galactosidase assay (A615 values) for quantification of interaction strength (n = 3 clones). Negative control is measured as interaction of CLDN16 bait vector with an empty prey vector (pPR3N). (B) Coimmunoprecipitation assay showing Trpv5 (Flag-Trpv5) interacts with phosphomimetic claudin-16 (GFP-Cldn16-T303E) in transfected HEK293 cells. Asterisk indicates that lane shows 10% of input amount as in other lanes.

Fig. 7.

TJ localization and ZO-1 interaction of phosphorylated claudin-16. (A) Confocal microscopy showing TJ localization of wildtype claudin-16 and apical membrane localization of phosphomimetic claudin-16 in transfected LLC-PK1 cells. The TJ was labeled with an antibody against ZO-1. (Scale bar: 10 μm.) (B) Coimmunoprecipitation assay showing that wildtype claudin-16 (GFP-Cldn16-WT) but not phosphomimetic claudin-16 (GFP-Cldn16-T303E) interacts with ZO-1 (Myc-ZO-1) in transfected HEK293 cells.

Fig. 8.

Claudin-16 phosphorylation levels in PTH1R KO mice. (A) Freshly isolated mouse distal tubular cells from Ksp-Cre/PTH1R^fl/fl mice were lysed in Laemmli buffer and immunoblotted against anti-CLDN16-P antibody to reveal claudin-16 phosphorylation levels. Anti-tubulin antibody was used for loading control (**P < 0.01; n = 3 animals). (B) Dual immunofluorescent staining of phosphorylated claudin-16 protein (CLDN16-P) with the DCT markers (parvalbumin and calbindin-D28K). (Scale bar: 20 μm.)