Claudin-2 is a typical tight junction protein of leaky epithelia that forms paracellular channels for small cations and water. Claudin-3 and claudin-1 are barrier formers and may interact with claudin-2. We aimed to investigate whether this interaction affects the permeability of claudin-2 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 claudin-2 and claudin-2/claudin-3. Furthermore, MDCK II/quinKO/claudin-2/claudin-1 cells were generated for comparison. Electrophysiological assays were performed to evaluate the function and properties of claudin-2 channels in these cell models. Cis- and trans-interaction of claudin-2 with claudin-1 or claudin-3 was assessed in MDCK II/quinKO cells by FRET and enrichment assays, respectively. At the tight junction, claudin-2 had a closer cis-proximity to claudin-1 than to claudin-3, but a stronger trans-interaction with the latter. In comparison to cells expressing claudin-2 alone, co-expression with claudin-3 (in both cell models) or claudin-1 (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 claudin-2 and those expressing claudin-2/claudin-3. In sum, interaction of the barrier-formers claudin-3 or - 1 with the channel-former claudin-2 reduces its ion but not its water permeability. 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.