Abstract
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.
Highlights
Epithelial barriers are essential for the survival of multicellular organisms and allow compartmentalization and controlled interactions between distinct environments (Marchiando et al, 2010, Turner, 2009)
We have defined the biophysical behavior of claudin-2-dependent single channel conductance events in both canine kidney and human intestinal epithelia
The data confirm that claudin-2 channels within tight junctions are driven passively by the transepithelial, apical to basolateral electrochemical gradient
Summary
Epithelial barriers are essential for the survival of multicellular organisms and allow compartmentalization and controlled interactions between distinct environments (Marchiando et al, 2010, Turner, 2009). Intestinal epithelial expression of the tight junction protein claudin-2, which increases paracellular Na+ conductance (Amasheh et al, 2002, Wada et al, 2013, Weber et al, 2010), is downregulated after the neonatal period (Holmes et al, 2006) but markedly upregulated in inflammatory and infectious enterocolitis and by several cytokines, including IL-13 (Heller et al, 2005).
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