PROJECT SUMMARY Cholestasis is a common manifestation of liver disease. Cholestasis often is due to disorders specifically affecting cholangiocytes, which play a major role in bile secretion. They are responsible for secretion of bicarbonate into bile and modulating the biliary contents of other constituents as well. The type III inositol trisphosphate receptor (InsP3R-3) is the primary intracellular calcium release channel in cholangiocytes and our previous studies have shown that apically-localized InsP3R-3 controls bicarbonate secretion. We also found that in most ductular forms of human cholestasis and in multiple animal models, there is loss of InsP3R-3 expression, underlying the importance of calcium homeostasis in normal cholangiocyte function and its dysregulation in cholestasis. We hypothesize that restoration of InsP3R-3 expression will ameliorate cholestasis and improve biliary bicarbonate secretion. Thus the long term objective of this grant is to understand the molecular mechanisms of InsP3R-3 regulation in normal and cholestatic liver and the therapeutic effect of restoration of InsP3R- 3 expression on liver function. To achieve this objective, the proposal will be implemented with the following specific aims: (1) The molecular factors governing the regulation of InsP3R-3 gene expression will be defined by studying the transcription factors (TFs) and microRNAs (miRs) that regulate the InsP3R-3 promoter and mRNA respectively; (2) The cellular mechanisms that direct InsP3R-3 to the subapical region of cholangiocytes including targeting sequences and interacting proteins that lead to such localization will be established; (3) Molecular mechanisms that lead to loss of InsP3R-3 expression and function in animal/human models of cholestasis (including InsP3R-3 KO mice and CRISPR/Cas9-InsP3R-3-null human cholangiocytes) will be determined and verified in human cholestatic liver. Further, we will investigate whether restoration of expression-specific TFs/miRs-anti-miRs in cholestasis results in improvement of disease. Together, the data derived from these studies will improve our understanding of the regulation of secretion in cholangiocytes, and have the potential to lead to design of novel therapeutic approaches for the treatment of cholestatic disorders.