Project Summary/Abstract Primary cilia are unique organelles that protrude from the cell membrane of almost all vertebrate cells. Defective primary cilia, caused by mutations in proteins required for cilia formation or signaling, can result in ciliopathies, pleiotropic diseases affecting multiple organs. In particular, Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most prevalent monogenic disease leading to end-stage renal disease (ESRD), is considered ciliopathy. The polycystin channel complex, formed by the transmembrane proteins PC-1 and PC-2, is abundantly expressed primary cilia, and mutations in PC-1 and PC-2 account for the vast majority of ADPKD. To date, we only have a limited functional understanding of how the polycystin complex commands the electric signaling of primary cilia via cation flux (K+, Na+, and Ca2+) due to technical challenges in characterizing channel activity of ciliary membranes. To better understand, I established ciliary patch-clamp recordings to measure the activity of endogenous polycystin channels directly from the ciliary membrane. I published in 2020 that the C- type lectin domain (CTL) of the PC-1 N-terminus plays a crucial role in polycystin activation, highlighting the indispensable participation of PC-1 subunits. In this proposal, using my established assays, I will determine how ADPKD-causing mutations within the N-terminus of PC-1 impair polycystin function. Further, I will establish ciliary patch-clamp recordings of primary cilia within kidney organoids, allowing me for the first time to functionally connect the molecular phenotype of impaired channel activity with the macroscopic phenotype of cyst formation. During the mentored K99 phase, I will characterize the functional impact of pathogenic variants within the PC-1 subunit using the ciliary patch-clamp recording. To understand the endogenous regulation of the polycystin complex, I will determine the specificity and potency for the cilia- enriched oxysterols to activate the polycystin complex in the cell or ciliary membrane. To complete this aim, I will receive further training in cilia biology in the Delling lab. In parallel, co-mentor Dr. Meyeon Park will guide me to understand the clinical significance of pathogenic mutants in PC-1. To expand knowledge of polycystin complex under more physiological conditions, I plan to measure electric signaling of primary cilia using kidney organoids during the R00 phase. Kidney organoids provide a powerful tool for understanding development and disease and finding new treatments and regenerative approaches. Characterizing ciliary ion channels in organoids will establish an essential milestone to understand the role of primary cilia in different segments of renal tubules and cyst development. During this time, the candidate will complete mentored training in the Department of Physiology in preparation for the independent R00 phase with the help of Dr. Benjamine S. Freedman at the University of Washingto...