Effect of claudin-1 or -3 expression on cation and water channel properties of claudin-2

Claudin-2 (Cldn2) is a typical tight junction protein of leaky epithelia that forms paracellular channels for small cations and water. Claudin-3 (Cldn3) and claudin-1 (Cldn1) are barrier formers and may interact with Cldn2. We aimed to investigate whether this interaction affects the permeability of Cldn2 channels to ions and/or water. To achieve this, two knockout kidney cell lines (MDCK C7/Cldn3KO and MDCK II/quinKO) were used to express Cldn2 and Cldn2/Cldn3. Furthermore, MDCK II/quinKO/Cldn2/Cldn1 cells were generated for comparison. Electrophysiological assays were performed to evaluate the function and properties of Cldn2 channels in these cell models. Cis- and trans-interaction of Cldn2 with Cldn1 or Cldn3 was assessed in MDCK II/quinKO cells by FRET and enrichment assays, respectively. At the tight junction, Cldn2 had a closer cis-proximity to Cldn1 than to Cldn3, but a stronger trans-interaction with the latter. In comparison to cells expressing Cldn2 alone, co-expression with Cldn3 (in both cell models) or Cldn1 (in MDCK II/quinKO cells) resulted in lower cation permeabilities without altering the Eisenman sequences. Other than ion permeability, water flux showed no differences between MDCK C7/Cldn3KO cells expressing Cldn2 and those co-expressing Cldn2/Cldn3. Based on these results, we propose a model in which Cldn2-Cldn1 cis- and Cldn2-Cldn3 trans-interaction leads to a mixture of homo-oligomeric Cldn2 and hetero-oligomeric Cldn2/Cldn1 or Cldn2/Cldn3 channels. The latter would have a pore center where charges are neutralized, by this impairing cation permeability while still allowing water to pass.