Structure, function, and modulation of claudin cation channels in the GI tract

ABSTRACT Intestinal diseases, including inflammatory bowel disease (IBD), celiac disease, and infectious colitis are associated with epithelial barrier dysfunction that contribute to diarrhea and nutrient malabsorption. Tight junctions seal spaces between epithelial cells and maintain barrier function by controlling paracellular flux. The claudin family of tight junction proteins is critical in defining the tight junction barrier to ions and small molecules. In the gastrointestinal tract, claudin-2 and -15 are particularly important in their role regulating paracellular sodium flux and their altered expression can contribute to intestinal disease development. Using a novel patch clamp technique, we demonstrated that claudin-2 and -15 form gated Na+ selective ion channels in the paracellular space. This is important because it demonstrates that claudins have properties similar to transmembrane ion channels. However, we have limited understanding of how these cation selective claudins contribute to charge and size selective paracellular pores, how these two claudins function singly or together to impact intestinal function, and how they impact disease processes. To address these questions, we built all-atom computer models for claudin-2 and -15 which allowed us to model both claudin structure and pore function. These models also helped us to identify several first-in-class claudin channel blockers that block the pore at low micromolar concentrations. We propose to use these unique and novel computational and small molecule inhibitor tools to investigate how claudin-2 and -15 channels control monovalent cation flux across the tight junction, and how they may differentially regulate cation transport in health and disease. In Aim 1, we will use our claudin-2 and -15 computer models and channel blockers to determine key molecular and structural features that dictate size and charge selectivity. In Aim 2, we will use our existing and new claudin channel blockers to define the individual and combined contributions of claudin-2 and -15 to normal intestinal physiology and disease presentation and development. We will determine the role of claudin-2 and -15 channels in Na+-coupled nutrient co-transport- mediated barrier regulation, mouse models of colitis, and in human IBD. Completion of this line of study is expected to contribute positively to human health by providing key insight into how these channels mediate nutrient absorption, contribute to diarrhea in the setting of colitis, and potentially aid in the development of novel therapies.