Cryptdins are a form of defensin released into the intestinal crypt lumen by specialized epithelial cells such as Paneth cells. These molecules exhibit antimicrobial activity and thus likely participate in defense of the crypt epithelial surface and perhaps in defense of the intestinal epithelial surface in general. We have generated pilot data indicating that these molecules can, by interacting with crypt apical membranes, alter the electrophysiological properties of crypt epithelial monolayers. Specifically purified cryptdin induces net electrogenic transport of ions across this epithelium associated with the introduction of an apical channel. These data are of particular interest since it is known that other species of defensin molecules can produce anion selective pores in artificial membranes. The data in hand are most consistent with cryptdin induction of electrogenic C1 transport- an event which protects mucosal surfaces by """"""""flushing"""""""" them with isotonic solution. In this project we will first characterize the nature of the transport event stimulated by cryptdins. Secondly, we will then use crypt epithelial monolayers with permeabilized basolateral membranes to better characterize the nature of the pore induced by cryptdins. Thirdly, we will examine fractions derived from purified cryptdin species in order to identify the specific molecule(s) in the crytdin fraction responsible for the effects on crypt epithelia. Lastly, we will use synthetic peptides to verify the peptide(s) species responsible for these effects on epithelial transport and then synthesize mutant peptide species to perform structure-function analyses. Such data should provide insights into cross-talk between Paneth cells and other crypt cells and yield information concerning how these cell type interact in defense to the crypt unit.
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