Defining single-channel paracellular (tight junction) conductances using nanotechnology

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PROJECT SUMMARY/ABSTRACT Epithelia and endothelia form barriers that separate the internal and external milieus and maintain isolated compartments within organisms. These barriers are sealed by intercellular tight junctions that are assembled over claudin protein polymer networks. Beyond forming the barrier, claudins also create size- and charge- selective paracellular channels that accommodate ions and water. Because of these divergent functions, individual claudins are classified as sealing or pore-forming. Studies in mice and humans demonstrate that mutations of sealing or pore-forming claudins are causes of heritable disorders. Even without mutation, regulated changes in claudin isoform expression contribute to disease pathogenesis. For example, intestinal epithelial claudin-2 expression is upregulated in colitis, and we have shown that claudin-2 channel inactivation by genetic or pharmacological approaches markedly attenuates experimental immune-mediated colitis. Until recently, claudin channels were thought of as fixed conduits that allow continuous paracellular flux. Our development of the trans-tight junction patch clamp allowed the paradigm-altering discovery that claudin-2 channels open and close dynamically to create quantal paracellular conductance events (Weber et al, eLife, 2015). This observation generated many new questions with fundamental, translational, and therapeutic impact. It has not, however, been possible to address these questions using the trans-tight junction patch clamp method, which measures only a single, very small, area of junction and has proven too labor-intensive and technically difficult for application beyond our proof-of-principle analyses. For example, it has not, been possible to determine whether all claudin channels are dynamic; if different claudins create channels with distinct biophysical properties, e.g., open probability or conductance event size; or how these characteristics can be modulated by cellular regulatory processes and pharmacologic agents. This exploratory grant proposal seeks to apply nanotechnology to analysis of claudin channel function by creating a nanochip populated by an array of individually addressable, nanopillar-mounted electrodes. After culture of epithelial cell monolayers on these chips, electrodes within lateral intercellular spaces, just beneath the tight junctions, can be used to evaluate channel conductances. This approach obviates the difficult process of patching a GΩ seal across the paracellular space between adjacent cells. By eliminating the patch pipette and making concurrent analysis of multiple junctions and channels within a single monolayer possible, the tight junction-sensing nanochip will overcome the main limitations of the trans-tight junction patch clamp. This novel, enabling technology will lead to new fundamental, paradigm-changing discoveries and, ultimately, knowledge and tools needed for development of agents that regulate cellular tight junction barriers in order to treat disorders of epithelial barrier function at diverse sites including the gut, kidneys, and lungs.