1.H.1 The Claudin Tight Junction (Claudin1) Family

Epithelia form boundaries of biological compartments, creating specialized absorptive and secretive surfaces such as the kidney tubules, the intestinal tract, and the mammary gland. The ability of epithelial cells to regulate absorption and secretion of essential ions such as sodium, chloride, calcium, and magnesium is critical for the maintenance of electrolyte balance (Van Itallie and Anderson, 2006). Ion transport across an epithelial layer can be either transcellular or paracellular (Shen et al., 2011). The transcellular pathway involves the movement of ions across the cytoplasm via plasma membrane channels, carriers, and exchangers (Muto et al., 2011). The paracellular pathway involves the movement of ions through the intercellular spaces between epithelial cells. The transmembrane proteins of tight junctions include claudins, junctional adhesion molecules (JAMs), occludin and tricellulin. Chiba et al. (2008) have provided an overview of these proteins, highlighting their roles and regulation, as well as their functional significance in human diseases. Sequence analysis of claudins has led to differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and non-classic claudins (11-13, 16, 18, 20-24), according to their degree of sequence similarity (Krause et al., 2008).  Claudins have been reviewed from structural/functional standpoints (Krause et al. 2015). All of the identified tight junction transmembrane proteins can be multiply phosphorylated, but only in a few cases are the consequences of phosphorylation at specific sites well characterized (Van Itallie and Anderson 2017). Passive solute transport across primary alveolar epithelial cell monolayers can be mediated by intercellular tight junctions (Kim et al. 2021). Claudins are not expressed exclusively by epithelial cells, but also by certain types of cells of mesodermal origin (Čužić et al. 2021). Multiscale modelling of claudin-based assemblies has been reported (Berselli et al. 2022).

The architecture of tight junctions can be conceptualized into compartments with the transmembrane barrier proteins (claudins, occludin, JAM-A, etc.), linked to peripheral scaffolding proteins (such as ZO-1, afadin, MAGI1, etc.) which are in turned linked to actin and microtubules through numerous linkers (cingulin, myosins, protein 4.1, etc.) (Van Itallie and Anderson 2014). Within this complex network are associated many signaling proteins that affect the barrier and broader cell functions. The PDZ domain is a commonly used motif to specifically link individual junction protein pairs. Van Itallie and Anderson 2014 reviewed some of the key proteins defining the tight junction as well as their detailed architecture and subcompartments. Claudins 1 and 3 can form homo- and heterophilic cis and trans interactions, and at least two different cis-interaction interfaces within claudin-3 homopolymers as well as within claudin-1/claudin-3 heteropolymers have been documented (Milatz et al. 2015). The disruption of the barrier between the endolymph and perilymph in the auditory system, caused by tight junction abnormalities, can affect the microenvironment of hair cells, and this can be the reason for one type of hearing loss. This topic has been reviewed (see below; Gao et al. 2023).

Two TJ protein families can be distinguished, claudins, comprising 27 members in mammals, and TJ-associated MARVEL proteins (TAMP), comprising occludin, tricellulin, and MarvelD3 (Krug et al. 2014). They are linked to a multitude of TJ-associated regulatory and scaffolding proteins. The major TJ proteins are classified according to the physiological role they play in enabling or preventing paracellular transport. Many TJ proteins have sealing functions (claudins 1, 3, 5, 11, 14, 19, and tricellulin). In contrast, a significant number of claudins form channels across TJs which feature selectivity for cations (claudins 2, 10b, and 15), anions (claudin-10a and -17), or are permeable to water (claudin-2). For several TJ proteins, function is yet unclear as their effects on epithelial barriers are inconsistent (claudins 4, 7, 8, 16, and occludin). TJs undergo physiological and pathophysiological regulation by altering protein composition or abundance. Major pathophysiological conditions which involve changes in TJ protein composition are (1) effects of pathogens binding to TJ proteins, (2) altered TJ protein composition during inflammation and infection, and (3) altered TJ protein expression in cancers (Krug et al. 2014). CLDNs6 and 9 are almost identical, differing only with respect to three extracellular residues, but a monoclonal antibody speicific for CLDN6, which is upregulated in cancers, has been isolated (Screnci et al. 2022).

The gatekeeper of the paracellular pathway is the tight junction, which is located at apical cell-cell interactions of adjacent epithelial cells. Three known inherited disorders, familial hypomagnesemia (Simon et al., 1999), hypertension (Wilson et al., 2001), and autosomal recessive deafness (Wilcox et al. 2001) have been linked to proteins that localize at the tight junction. Transmembrane proteins of tight junctions include claudins, junctional adhesion molecules (JAMS), occludin and tricellulin. The cytoplasmic scaffolding proteins include Z0-1, -2 and -3 (Hartsock and Nelson, 2008).Their study has led to insights into the molecular nature of tight junctions (Chiba et al., 2008). Neurological diseases (Bednarczyk and Lukasiuk, 2011) and renal diseases (Li et al., 2011) have been reviewed. High concentrations (>200 μM) of Zn²⁺ can affect TJ integrity in a polarized manner. Thus, the basolateral addition of Zn²⁺ leads to reversible TJ opening with pore paths of r ∼ 2 nm or more, depending on the Zn²⁺ concentration.  Zn²⁺-induced paracelluar channels favour efflux especially for macromolecules (Xiao et al. 2018). Even bacterial outer membrane vesicles have been shown to pass through 'leaky' junctions (Jones et al. 2020).

Tight junctions of epithelial cells exclude macromolecules but allows permeation of ions. It has not been clear whether this ion-conducting property is mediated by aqueous pores or by ion channels. To investigate the permeability properties of the tight junction, Tang & Goodenough (2003) developed paracellular ion flux assays for four major extracellular ions, Na⁺, Cl^-, Ca²⁺, and Mg²⁺. Tight junctions share biophysical properties with conventional ion channels, including size and charge selectivity, dependency of permeability on ion concentration, competition between permeant molecules, anomalous mole-fraction effects, and sensitivity to pH. Their results support the hypothesis that discrete ion channels are present at the tight junction. Unlike conventional ion channels, which mediate ion transport across lipid bilayers, the tight junction channels must orient parallel to the plane of the plasma membranes to support paracellular ion movements. This new class of paracellular-tight junction channels facilitates the transport of ions between separate extracellular compartments (Balkovetz, 2009). Claudin-2 forms highly cation-selective paracellular pores (Yu, 2009). The basis of this charge selectivity is likely to be the presence of a negatively charged binding site within the lumen of the pore.  Paracellin-1 may be a Mg²⁺ transporter (Brandao et al. 2012).

Heterotypic (head-to-head) binding between different claudin isoforms plays a role in regulating paracellular permeability. Claudin-3 and claudin-4 do not heterotypically interact despite having highly conserved extracellular loop (EL) domains (Daugherty et al., 2007). Claudin-1 and -5, which are heterotypically compatible with claudin-3, do not bind to claudin-4. In contrast, claudin-4 chimeras containing either the first EL domain or the second EL domain of claudin-3, do bind to claudin-1, claudin-3, and claudin-5. Moreover, a single point mutation in the first extracellular loop domain of claudin-3, converting Asn⁴⁴ to the corresponding amino acid in claudin-4 (Thr) produced a claudin capable of heterotypic binding to claudin-4 while still retaining the ability to bind to claudin-1 and -5. Thus, control of heterotypic claudin-claudin interactions is sensitive to small changes in the EL domains (Daugherty et al., 2007). Claudin-1, - 3, - 4, and - 7 are expressed mainly in the lateral plasmamembrane of serous glandular cells. In the ducts, claudin-1, - 4, and - 7 were detected at the basal cell layer; claudin-7 was found at the lateral cytomembrane (Stoeckelhuber et al. 2023).

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16 (Hou et al., 2007). Claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Claudin-16-deficient mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Claudin-16 apparently forms a non-selective paracellular cation channel, rather than a selective Mg²⁺/Ca²⁺channel as previously proposed (Hou et al., 2007). Claudin-16 increases transepithelial electrical resistance and transepithelial magnesium transport (Ikari et al., 2008). The activation of polyvalent extracellular cation (Ca²⁺)-sensing receptor (CaSR; P41180) decreased the resistance and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP. Activation of CaSR may thus decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosomes and a consequent decrease in magnesium reabsorption (Ikari et al., 2008).

Claudins comprise the primary constituents of tight junctions and determine paracellular permeability. Ion selectivity of the paracellular conductance is a complex function of claudin subtype and cellular context (Hou et al., 2007). These 4 TMS proteins have been characterized from structural standpoints and may have arisen from an early intragenic duplication event (Hua et al., 2003).  There are 27 claudin paralogues in mice and humans (Mineta et al. 2011).  Permselective paracellular claudin channels are specific for certain ions and non-ionic solutes. Recent studies using claudin knockout mice revealed that the loss of claudins' specific paracellular barrier and/or channel functions affects particular biological functions and leads to pathological states (Tamura and Tsukita 2014). Palmitoylation of claudins is required for efficient tight-junction localization (Van Itallie et al. 2005). Human peritoneal tight junctions, transporters and channels are expressed differentially in health and kidney failure, and are described by Levai et al. 2023.

As reviewed by Angelow et al. (2007;2008), the structure of claudin-based paracellular pores is largely unknown, but it is probably composed of homo- and hetero-typic claudin digomers. Both the proteins involved and the cell type determine the selectivity of paracellular transport. Claudins 2, 106 and 15 act preferentially as cation pores while claudins 10a and 7 are the only claudins that have significant anion pore properties (Angelow et al., 2008). However, claudins 4 and 7 have been reported to act as cation pores in MDCK II cells but as anion pores in LLC-PK 1 cells (Hou et al., 2006). They can pass neutral as well as charged small molecules. Their pore diameters are 8-15 Å. The first extracellular loop may line the paracellular pathways and determine the charge selectivity, but the C-terminal tail, which is modified by phosphorylation and palmitoylation and interacts with cytoskeletal proteins, may also play a role.

Claudin-2 pores are narrow, fluid filled, and cation selective (Yu et al., 2009). Charge selectivity is mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity. Post-translational modifications, PTMs of TJ proteins directly contributes to epithelial barrier changes in permeability to ions and macromolecules (Reiche and Huber 2020).

The control of claudin assembly into tight junctions requires a complex interplay between several classes of claudins, other transmembrane proteins and scaffold proteins (Findley and Koval, 2009). Claudins are also subject to regulation by post-translational modifications including phosphorylation and palmitoylation. Several human diseases have been linked to claudin mutations. Roles for claudins in regulating cell phenotype and growth control suggest a multifaceted role for claudins in regulation of cells beyond serving as a simple structural element of tight junctions. Claudins (Cldns) promote the formation of either barriers or ion-selective channels at the interface between two facing cells, across the paracellular space (Berselli et al. 2022). Multiple Cldn subunits form complexes that include cis- (intracellular) interactions along the membrane of a single cell and trans- (intercellular) interactions across adjacent cells.  The recent implementation of computer-based techniques contributed to the elucidation of Cldn properties. Molecular dynamics simulations and docking calculations were extensively used to refine the first Cldn multimeric model postulated from the crystal structure of Cldn15, and contributed to the introduction of a novel alternative arrangement. Both these multimeric assemblies were found to account for the physiological properties of some family members. Berselli et al. 2022 illustrated the major findings on Cldn-based systems that were achieved by using state-of-the-art computational methodologies.

Epithelial transport relies on the proper function and regulation of the tight junction (TJ); otherwise, uncontrolled paracellular leakage of solutes and water would occur. They also act as a fence against mixing of membrane proteins of the apical and basolateral side. The proteins determining paracellular transport consist of four transmembrane regions, intracellular N and C terminals, one intracellular and two extracellular loops (ECLs). The ECLs interact laterally and with counterparts of the neighboring cell and thereby achieve a general sealing function. Two TJ protein families can be distinguished, claudins, comprising 27 members in mammals, and TJ-associated MARVEL proteins (TAMP), comprising occludin, tricellulin, and MarvelD3. They are linked to a multitude of TJ- associated regulatory and scaffolding proteins (Günzel and Fromm 2012). The major TJ proteins are classified according to the physiological role they play in enabling or preventing paracellular transport. Many TJ proteins have sealing functions (claudins 1, 3, 5, 11, 14, 19, and tricellulin). In contrast, a significant number of claudins form channels across TJs which feature selectivity for cations (claudins 2, 10b, and 15), anions (claudin-10a and -17), or are permeable to water (claudin-2). For several TJ proteins, their functions are unclear as their effects on epithelial barriers are inconsistent (claudins 4, 7, 8, 16, and occludin). TJs undergo physiological and pathophysiological regulation by altering protein composition or abundance. Major pathophysiological conditions which involve changes in TJ protein composition are (1) effects of pathogens binding to TJ proteins, (2) altered TJ protein composition during inflammation and infection, and (3) altered TJ protein expression in cancers (Günzel and Fromm 2012). 

The electric property of claudin pertains to two important organ functions: the renal and sensorineural functions. The kidney consists of three major segments of epithelial tubules with different paracellular permeabilities: the proximal tubule (PT), the thick acending limb of Henle's loop (TALH) and the collecting duct (CD). Claudins act as ion channels allowing selective permeation of Na⁺ in the PT, Ca²⁺ and Mg²⁺ in the TALH and Cl^- in the CD. The inner ear, on the other hand, expresses claudins as a barrier to block K⁺ permeation between endolymph and perilymph. The permeability properties of claudins in different organs can be attributed to claudin interactions within the cell membrane and between neighboring cells. The first extracellular loop of claudins contains determinants of paracellular ionic permeability (Hou 2013).

The thick ascending limb (TAL) of Henle's loop drives paracellular Na⁺, Ca²⁺, and Mg²⁺ reabsorption via the tight junction (TJ). The TJ is composed of claudins with two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. Claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to disturbances of renal ion homeostasis. Milatz et al. 2017 showed that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by Cldn16 favor Mg²⁺ over Na⁺; (iii) Cldn10b does not interact with other TAL claudins, whereas Cldn3 and Cldn16 can interact with Cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. Milatz et al. 2017 suggested the existence of at least two spatially distinct types of paracellular channels in TAL: a Cldn10b-based channel for monovalent cations such as Na⁺ and a spatially distinct site for reabsorption of divalent cations such as Ca²⁺ and Mg²⁺. 

Tight junctions (TJ) play a central role in the homeostasis of epithelial and endothelial tissues, by providing a semipermeable barrier to ions and solutes, by contributing to the maintenance of cell polarity, and by functioning as signaling platforms. TJ are associated with the actomyosin and microtubule cytoskeletons, and the crosstalk with the cytoskeleton is fundamental for junction biogenesis and physiology. TJ are spatially and functionally connected to adherens junctions (AJ), which are essential for the maintenance of tissue integrity. Mechano-sensing and mechano-transduction properties of several AJ proteins have been characterized during the last decade. Citi 2019 reviews how mechanical forces act on TJ and their proteins, how TJ control the mechanical properties of cells and tissues, and what the underlying molecular mechanisms are. Interactions among the adherence junctional proteins is influenced by phosphorylation (Wang et al. 2019).

Tight junctions act as a barrier between epithelial cells to limit the transport of paracellular substances, which is a required function in various tissues to sequestrate diverse microenvironments and maintain a normal physiological state (Gao et al. 2023). Tight junctions are complexes that contain various proteins, like transmembrane proteins, scaffolding proteins, signaling proteins, etc. Defects in these tight junction-related proteins can lead to hearing loss which is also recapitulated in model organisms. The disruption of the barrier between the endolymph and perilymph caused by tight junction abnormalities affect the microenvironment of hair cells, and this may be the reason for some types of hearing loss. Besides their functions as a typical barrier and channel, tight junctions are also involved in many signaling networks to regulate gene expression, cell proliferation, and differentiation. Gao et al. 2023 summarized the structures, localization, and signaling pathways of hearing-related tight junction proteins and their potential contributions to the hearing disorder.

The paracellular transport reactions proposed to be catalyzed by claudinins are:

Ions (Lumen) Ions (Tissues).

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