Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex

Abstract

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl − blocker, whereas CLDN16 functioned as a Na + channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl − permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC. IntroductionThe human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC; OMIM 248250) is characterized by progressive renal Mg 2+ and Ca 2+ wasting leading to impaired renal function and chronic renal failure. FHHNC has been genetically linked to mutations in the gene of claudin-16 (CLDN16, also known as paracellin-1) (1) and more recently to CLDN19 (2). The claudins comprise a 22-gene family that encodes essential structural proteins of the tight junction (TJ), which are the principal regulators of paracellular permeability. In vitro studies have shown that ion selectivity of the paracellular conductance (reviewed in ref.3) is a complex function of claudin subtype and cellular context (4, 5).In vitro analyses using cultured cell models show that CLDN16 plays a key role in maintaining the cation selectivity of the TJ and forms a nonselective paracellular cation channel (4). This hypothesis of a nonselective paracellular cation channel is supported by (a) a clinical study to correlate the cellular functions of CLDN16 mutations identified in FHHNC to the phenotypes of FHHNC patients, with a special focus on the progression of renal failure (6), and by (b) our mouse models using transgenic RNAi depletion of CLDN16 (7). Without CLDN16 expression in the kidney, TJs in the thick ascending limb (TAL) of the nephron lose cation selectivity, leading to the dissipation of the lumen-positive potential with a concomitant loss of the driving force for Mg 2+ reabsorption (7). While targeted deletion of CLDN19 in mice initially focused on its

Biology of claudins

“…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 ).…”

“…Indeed, claudins-16 and -19 completely colocalize in the TALH and thin ascending limbs of Henle (6,61). Overexpression of claudin-19 alone has yielded conflicting results (6,45). However, when claudins-16 and -19 are coexpressed, they interact in a yeast two-hybrid system, suggesting that they may form heteromultimers (45).…”

“…Overexpression of claudin-19 alone has yielded conflicting results (6,45). However, when claudins-16 and -19 are coexpressed, they interact in a yeast two-hybrid system, suggesting that they may form heteromultimers (45). Coexpression of claudins-16 and -19 increased P Na /P Cl more than either one alone, suggesting that this may be important for generating the dilution potential in the TALH.…”

Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium

“…In the kidney, CLDN19 is also exclusive to the TAL (15). In vitro, CLDN16 and CLDN19 interact and form a cation-selective TJ paracellular channel (18). On the basis of these observations, we hypothesized that the CLDN19-deficient animals will phenocopy the CLDN16 knockdown (KD) and that CLDN16-CLDN19 interaction is a critical component of paracellular cation selectivity in the TAL.…”

Clinical utility gene card for: familial hypomagnesemia with hypercalciuria and nephrocalcinosis with/without severe ocular involvement

“…1,2 These genes encode the tight-junction proteins claudin-16 (paracellin-1) and claudin-19, respectively, which are important for the paracellular reabsorption of calcium and magnesium in the thick ascending limb of Henle's loop. 3 Claudin-19 is also expressed in the retinal epithelium. FHHNC is characterized by excessive urinary losses of magnesium and calcium, nephrocalcinosis, kidney stones in about 1/3 of cases and progressive renal failure.…”