PROJECT SUMMARY The intestinal epithelium relies on extensive cell-cell signaling to maintain a tight balance between absorptive and secretory activity, but the intricacies of this signaling remain uncharacterized. We recently discovered that cells infected with rotavirus, the most common cause of life-threatening diarrhea worldwide, release hundreds of pulses of adenosine diphosphate (ADP) causing intercellular calcium waves that dysregulate uninfected cells. Blocking the ADP receptor P2Y1 significantly reduces diarrhea severity in rotavirus-infected mice, suggesting that this signaling is integral in regulating secretory activity. While the mechanism through which rotavirus triggers the release of ADP has not been confirmed, our preliminary data show it relies on activation of the volume- regulated anion channel (VRAC). VRAC is increasingly recognized as a conduit for paracrine signals associated with a variety of intracellular stressors, but it has yet to be studied within the epithelium. Further, the mechanisms regulating its activation in any context remain poorly understood. Therefore, the objective of this proposal is to use rotavirus as a tool to study the role and activation of VRAC in the intestinal epithelium. Given that rotavirus-induced intercellular calcium waves are inhibited by knock down of rotavirus non-structural protein 4 (NSP4), a Ca2+-conducting channel, my central hypothesis is that NSP4-induced elevations of cytosolic Ca2+ activate host pathways that trigger ADP release through VRAC, amplifying secretory activity throughout the epithelium. I will test this hypothesis by using long-term live calcium imaging in conjunction with novel fluorescent Rho biosensors and CRISPR-Cas9 based gene editing to [Aim 1] determine the intracellular pathway mediating VRAC activation in RV-infected cells. Given ADP release is reduced upon pharmacological inhibition of Rho kinase or knockdown of the calcium-augmenting rotavirus protein NSP4, we expect both calcium and Rho are involved in VRAC activation. Using both human intestinal enteroids and the mouse model of rotavirus diarrhea, I will [Aim 2] determine the effect of VRAC activation on secretory activity in the intestinal epithelium. This work will identify novel therapeutic targets for the treatment of secretory diarrhea, a leading cause of mortality among children worldwide. Furthermore, our findings will address gaps in knowledge surrounding VRAC activation and its role in paracrine signaling. Given that VRAC has been implicated in an increasing variety of pathological processes, using this clinically relevant approach will yield information translatable to both human health and other scientific disciplines.