Claudin‐4 Disrupts Claudin‐2 Anchoring and Pore Function via Cis, but not Trans, Interactions at the Tight Junction

Tight junction strands are composed of claudin (cldn) proteins. The consensus view is that cldns either form pores that mediate paracellular flux or form seals that prevent such flux. An alternative view is that cldns that reduce paracellular flux do so by competitively disrupting pore‐forming cldn function. Both trans‐ and cis‐interactions between similar (homotypic) and dissimilar (heterotypic) cldns have been implicated in strand assembly and anchoring at the tight junction. We sought to test the cldn competition model and define the topology of cldn‐cldn interactions involved in cldn2 anchoring. mEGFP‐cldn‐2 expression in monolayers of high resistance MDCK I cells, which lack endogenous cldn2, reduced transepithelial electrical resistance (TER) by 38%±3%. Conversely, mCh‐cldn4 expression increased TER of mEGFP‐cldn‐2 expressing monolayers. These data indicate that cldn4 can oppose cldn2‐dependent paracellular pore function, but do not distinguish between pore‐seal and competition models of cldn function. The latter is, however, favored by the observation that cldn4 knockout does not significantly affect TER of MDCK I monolayers. Fluorescence recovery after photobleaching (FRAP) analyses demonstrated an mEGFP‐cldn2 mobile fraction (Mf) of 18%±3%, while mCherry‐cldn4 displayed a much higher Mf of 44%±4%. When mCherry‐cldn4 was expressed with mEGFP‐cldn2 at symmetric tight junctions, mEGFP‐cldn2 FRAP increased markedly, with an overall Mf of 35%±3%. This suggests that cldn4 increases TER by destabilizing cldn2 at the tight junction. Cldn2 stabilization at the tight junction did not require cldn2‐cldn2 trans‐interactions, as the mEGFP‐cldn2 Mf was similar at asymmetric junctions between cells expressing mEGFP‐cldn2 and non‐transfected MDCK I cells (23%±4%). Further, cldn4‐mediated cldn2 destabilization was not dependent on cldn4‐cldn2 trans‐interactions as demonstrated by similar mEGFP‐cldn2 Mf at asymmetric junctions between cells expressing mEGFP‐cldn2 and those expressing mCherry‐cldn4 (25%±3%). Cldn4 therefore modifies cldn2 anchoring at the tight junction via cis‐interactions within individual cells. These data support the cldn competition model and suggest that cldn4 increases TER by disrupting cldn2‐cldn2 cis‐interactions that are required for anchoring and pore function. Support or Funding Information DK061931