Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells

Abstract

Tight junctions seal the paracellular pathway of epithelia but, in leaky tissues, also exhibit specific permeability. In order to characterize the contribution of claudin-2 to barrier and permeability properties of the tight junction in detail, we studied two strains of Madin-Darby canine kidney cells(MDCK-C7 and MDCK-C11) with different tight junctional permeabilities. Monolayers of C7 cells exhibited a high transepithelial resistance (>1 kΩ cm2), compared with C11 cells (<100 Ωcm2). Genuine expression of claudin-1 and claudin-2, but not of occludin or claudin-3, was reciprocal to transepithelial resistance. However,confocal microscopy revealed a marked subjunctional localization of claudin-1 in C11 cells, indicating that claudin-1 is not functionally related to the low tight junctional resistance of C11 cells. Strain MDCK-C7, which endogenously does not express junctional claudin-2,was transfected with claudin-2 cDNA. In transfected cells, but not in vector controls, the protein was detected in colocalization with junctional occludin by means of immunohistochemical analyses. Overexpression of claudin-2 in the originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher paracellular conductivity and relative ion permeabilities of Na+≡1, K+=1.02, NMDG+=0.79,choline+=0.71, Cl-=0.12, Br-=0.10 (vector control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were not changed. Hence, with regular Ringer's, Na+ conductivity was 0.2 mS cm-2 in vector controls and 1.7 mS cm-2 in claudin-2-transfected cells, while Cl- conductivity was 0.2 mS cm-2 in both cells. Thus, presence of junctional claudin-2 causes the formation of cation-selective channels sufficient to transform a `tight'tight junction into a leaky one.

Cingulin Regulates Claudin-2 Expression and Cell Proliferation through the Small GTPase RhoA

“…However, our cingulin-depleted clones were obtained from MDCKII cells, which already express claudin-2, and have a relatively low TER. Cingulin-depleted monolayers showed a slight decrease in TER values at 4 -8 h after start of junction assembly by the calcium switch, consistent with the notion that increased claudin-2 levels correlate with lower TER, due to increased permeability to cations (Amasheh et al, 2002). However, this decrease in TER was not statistically significant, and at steady-state the barrier function of cingulin-depleted clones was similar to WT cells.…”

Tricellulin Forms a Barrier to Macromolecules in Tricellular Tight Junctions without Affecting Ion Permeability

“…), indicating that charge preference was not affected by TRIC-a ( Figure 5B). Especially for the TRa-4 clone this underlines that the change in claudin-2 expression had no functional relevance, because a change in Na ϩ permeability would be expected after elevated expression of properly located claudin-2, because claudin-2 is known to form specific paracellular channels for small cations Amasheh et al, 2002).…”

“…Single-ion permeabilities were determined from dilution or bi-ionic potentials and the Goldman-Hodgkin-Katz equation as reported previously (Amasheh et al, 2002;Gü nzel et al, 2009;Yu et al;. Briefly, NaCl dilution potentials were measured by switching one hemichamber to a solution containing a reduced concentration of NaCl and all other components identical to standard Ringer's.…”

Identification and functional clustering of global gene expression differences between age-related cataract and clear human lenses and aged human lenses

“…Another gene included in this category is claudin, a component of tight junction filaments capable of interacting adhesively with complementary molecules on adjacent epithelial cells (Gonzalez-Mariscal et al, 2003). Recent studies have found that over-expression of claudin-2 induces cation-selective channels in tight junctions of epithelial cells resulting in increased ion permeability (Amasheh et al, 2002). Other genes include supervillin, an F-actin bundling plasma membrane protein that contains functional nuclear localization signals (Wulfkuhle et al, 1999), bamacan, a chondroitin sulphate proteoglycan that abounds in basement membranes and is thought to be involved in the control of cell growth and transformation (Ghiselli et al, 1999) and osteonectin which has previously been demonstrated to be increased in human age-related cataracts (Kantorow et al, 2000).…”