NSP4 stimulated ion channels and age-dependent diarrhea

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DESCRIPTION (provided by applicant): Rotaviruses are a major cause of life-threatening diarrhea in infants and children worldwide. Following viral infection, diarrhea is seen associated with pathophysiological changes in mucosal fluid and electrolyte balance. My group has focused on defining a new pathophysiological component to diarrhea. We have shown that a rotaviral non-structural protein called NSP4 induces diarrhea in both normal and cystic fibrosis mouse pups accompanied by calcium-sensitive chloride secretory current generation by gastrointestinal mucosa. Neither diarrhea nor anion secretion occur in adult mice. At the sub-cellular level, NSP4 causes phospholipase C sensitive intracellular calcium (Ca2+)i mobilization and calcium-sensitive halide influx into mucosal crypts. NSP4-induced (Ca2+)i mobilization (our assay for receptor occupancy) is not age-dependent. Thus, we hypothesize that NSP4 activates and age-dependent calcium-sensitive chloride channel in pup mucosa causing chloride secretion, and secretory diarrhea. We propose studies in native cells to identify and characterize the electrophysiological and pharmacological properties of the chloride channel, and thus unequivocally demonstrate a role for this conductance in NSP4 mediated age-dependent cellular halide influx. We intend to identify the cellular signaling mechanisms coupling NSP4 mediated changes in (Ca2+)i to this conductance. These mechanistic studies may identify novel targets for pharmacological intervention with clear clinical relevance. These goals will provide the cellular basis for the age-dependent secretory diarrhea and may identify a molecular target for rotaviral-induced transepithelial anion secretion. They will also translate facts established for the biophysics of calcium-activated chloride channel expression in cultured epithelial cell-lines into the fields of clinical medicine and disease. In doing so, our results will provide an excellent possibility for development of new therapies for rotaviral gastroenteritis, and for other infectious diseases in children where altered mucosal (Ca2+)i homeostasis occurs.