Histochem Cell Biol (2008) 130:55–70 TJs as permeability barrier One of the key functions of tight epithelia is the creation of a diVusion barrier by tight control of the transcellular and paracellular pathways. This is acomplished by asymmetri- cal distribution of transporters and channels for the trans- cellular route, and on the other side by regulation of diVusion through the paracellular space via integral TJ pro- teins (Turner 2006). Hereby, certain claudins participate in the formation of ion-selective pores: available data suggest that TJs on the one hand are permeable to small noncharged solutes up to a size of 0.43–0.45 nm in radius as shown by the use of the membrane-impermeant polyethylene glycol as a tracer, but investigations also showed that pores within the TJs are capable of discriminating between ion charge and size, which both are additionally dependent on concen- tration and pH (Schneeberger and Lynch 2004; van Bree et al. 1988). The two extracellular loops of occludin consist solely of uncharched amino acid side chains (Fig. 3a) represented mainly by an unusually high content of tyrosine and glycine (Furuse et al. 1993) so that a direct role for occludin in con- ferring charge selectivity is doubtful. However, occludin could increase electrical resistance by interaction with the extracellular domains of the diVerently charged residues of the diVerent claudins (see below), since ablation of occlu- din is not without consequence despite the fact that the pro- tein does not appear to be required for TJ strand formation. Occludin-null mice express a complex phenotype, which is, however, not manifested by structural or functional TJ abnormalities: in occludin ¡ ¡ mice, TJs were not aVected / morphologically, and electrophysiological barrier function of intestinal epithelium was normal. However, histopa- thological abnormalities were detected in several tissues, i.e., chronic inXammation and hyperplasia of the gastric epithelium, calciWcation in the brain, testicular atrophy, the loss of cytoplasmic granules in striated duct cells of the salivary gland as well as the thinning of the compact bone, pointing to complex functions of the occludin protein (Saitou et al. 2000). Role of claudin proteins in formation of a TJ permeability barrier The lack of charged amino acid residues within the extra- cellular loops of occludin segregates occludin from the candidates contributing directly to the formation of ion- selective pores. The diVerent claudins whose extracellular loops span a wide range of pKi on the contrary appear to be excellent candidates to mediate ion selectivity in TJ perme- ability (Fig. 3b). Major insight into the distinct charge selectivity of the claudins came from the work of Furuse 59 et al. (2001), who could relate the diVerent levels of resis- tance between MDCK I cells (TER > 10,000 cm2) and MDCK II cells (TER <200 cm2) to a diVerential claudin isoform expression, while MDCK I cells express claudin-1 and -4 but not claudin-2, MDCK II cells additionally express claudin-2, which seems to mediate higher ion per- meability by pore formation/inducing strand breaks. Nota- bly, transfection of the tight MDCK I with claudin-2 cDNA resulted in 20-fold decline in TER but not in the number of TJ strands. A wide range of further transfection and mutagenesis experiments introducing or ablating diVerent claudins has been performed to date, and been partially helpful to eluci- date the conductance conferring properties of the resulting pores (Matter and Balda 2003b). These informative obser- vations must however be viewed with some caution because transfection and site-directed mutagenesis may lead to more than a change in net charge as for example an altered molecular conformation, or altogether the introduc- tion of an unusual claudin protein could lead to disturbance of strand organisation within native strands. By the con- struction of chimera, Colegio et al. (2003) were moreover able to attribute ion selectivity of the claudins to distinct stretches within the primary amino acid sequence of the extracellular loops leading to alterations in TER and charge selectivity in transfection experiments. Taken together, available data strongly suggest a key role for the extracellu- lar loops of the diverse claudins in the formation of ion selective pores within the TJ strands. Role of occludin in formation of a TJ permeability barrier The role of occludin in TJs and a putative contribution to building up the TJ’s ion selectivity (the ion pores) is unclear; it has been demonstrated that overexpression of occludin in MDCK cells reduces ion permeability but in the same time was shown to increase the transepithelial Xux of the uncharged compound mannitol (Balda et al. 1996). Fur- ther observations then implicated that occludin may be a target for receptor-initiated signalling involving the Rho family of GTPases, and that by an additional not-yet-under- stood mechanism, occludin phosphorylation may regulate TJ permeability independently of cytoskeletal activity (Hir- ase et al. 2001). Some indirect evidence for a role of occludin in strength- ening barrier properties arose from heterologous expression studies: when expressed in L-Wbroblasts that lack endoge- nous cadherin-based adherens junctions and ZO-1, little adhesiveness was measured (Kubota et al. 1999). However, when overexpressed in Wbroblasts, which do have well developed ZO-1-containing adherens-like junctions (NRK- 123