Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation.
Epithelial cells form layers that line the inner surface of the gut, lungs and other organs. They act as barriers to control the movement of water, ions and small molecules between internal compartments within the body and the external environment. Some substances are transported across these barriers by passing through individual epithelial cells, but others pass through the spaces between adjacent cells. These spaces are sealed by tight junctions. If the tight junctions do not work properly, it can cause problems with regulating the movement of molecules across epithelial-lined surfaces. This in turn can contribute to diseases in humans, including inflammatory bowel disease and chronic kidney disease.
Proteins called claudins form channels that only allow certain molecules to pass through tight junctions. One member of this family, called claudin-2, allows sodium ions and other small positively charged ions to cross between adjacent cells. However, it is not clear how these channels work, largely due to the absence of appropriate tools to study this process. Here, Weber et al. adapted a technique called patch clamping to study the behavior of individual claudin-2 channels in the tight junctions between mammalian epithelial cells.
Weber et al. found that claudin-2 allows positively charged ions to move across a tight junction in short bursts rather than in a steady stream as had been suggested by previous work. These bursts typically begin and end in less than a millisecond. Further experiments revealed that claudin-2 channels have several states; in one state the channel is fully open, in another the channel is firmly closed, and in the third state the channel is temporarily closed but primed to open.
Further experiments show that mutations in the gene that encodes claudin-2 or drugs that inhibit claudin-2's function alter the open and closed behaviors of these trans-tight junction channels. The technique developed by Weber et al. will enable researchers to understand how channel proteins at tight junctions assemble and operate. Such studies may lead to the development of drugs that can alter the activity of these channels to treat particular diseases.