Multicellular organisms are separated from the external environment by a layer of epithelial cells whose integrity is maintained by intercellular junctional complexes composed of tight junctions, adherens junctions, and desmosomes, whereas gap junctions provide for intercellular communication. The aim of this review is to present an updated overview of recent developments in the area of tight junction biology. In a relatively short time, our knowledge of the tight junction has evolved from a relatively simple view of it being a permeability barrier in the paracellular space and a fence in the plane of the plasma membrane to one of it acting as a multicomponent, multifunctional complex that is involved in regulating numerous and diverse cell functions. A group of integral membrane proteins-occludin, claudins, and junction adhesion molecules-interact with an increasingly complex array of tight junction plaque proteins not only to regulate paracellular solute and water flux but also to integrate such diverse processes as gene transcription, tumor suppression, cell proliferation, and cell polarity.

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

The tight junction (TJ) is a dynamic structure that is controlled, in part, by the activity of the cytoskeleton. It has become abundantly clear that, in the presence of Ca2+, assembly of the TJ is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation of an organized network of TJ elements, the composition of which remains unknown. The TJ functions both as a barrier between two fluid compartments and, to a lesser extent, as a fence between apical and basolateral membrane domains. To meet the many physiological and pathological challenges to which epithelia and endothelia are subjected, the TJ must be capable of a rapid and coordinated response, which depends on complex regulatory mechanisms. The precise characterization of the mechanisms involved in the assembly and regulation of the TJ is an area of current active investigation. However, until the biochemical composition of this structure has been defined and its gene identified, the TJ will continue to be an elusive yet tantalizing challenge to the cell biologist.

Epithelial tight junctions form a barrier against passive paracellular flux. This barrier is regulated by complex physiologic and pathophysiologic signals that acutely fine-tune tight junction permeability. Although actomyosin contraction and myosin light chain phosphorylation are clearly involved in some forms of tight junction regulation, the contributions of other signaling events and the role of myosin light chain phosphorylation in this response are poorly understood. Here we ask if activation of myosin light chain kinase alone is sufficient to induce downstream tight junction regulation. We use a confluent polarized intestinal epithelial cell model system in which constitutively active myosin light chain kinase, tMLCK, is expressed using an inducible promoter. tMLCK expression increases myosin light chain phosphorylation, reorganizes perijunctional F-actin, and increases tight junction permeability. TJ proteins ZO-1 and occludin are markedly redistributed, morphologically and biochemically, but effects on claudin-1 and claudin-2 are limited. tMLCK inhibition prevents changes in barrier function and tight junction organization induced by tMLCK expression, suggesting that these events both require myosin light chain phosphorylation. We conclude that myosin light chain phosphorylation alone is sufficient to induce tight junction regulation and provide new insights into the molecular mechanisms that mediate this regulation.