The Impact of ADPKD-Causing PKD1 Nonsense Variants on Polycystin Channel Function in the Primary Cilia

Project: Research project

Project Details


Autosomal dominant polycystic kidney disease (ADPKD) causes fatal progressive kidney failure and is commonly caused by variants in the PKD1 and PKD2 genes, which encode for two putative ion channel subunits polycystin-1 and polycystin-2. Despite our strong understanding of the genetic basis of ADPKD, we still do not know how variants in PKD1 and PKD2 impact their putative function as ion channels1,2. This basic question has remained outstanding for the past 15 years because the polycystins localize to the enigmatic primary cilium—a tiny antenna-like organelle that requires innovative tools to study. To this end, we have developed and deployed novel tools to study cilia channels which yield quantifiable results in real-time and at super resolution, thereby providing the most accurate description to date of ADPKD variants on ciliary ion channel function. We have published the first and highest resolution (3.0 Ȧ) structure of polycystin-2 ion channel using single-particle cryo-EM, which has provided a much-needed structural context for ADPKD-causing variants in the PKD2 gene. However, all structures available have been solved as a homotetramer, without the contribution of polycystin-1. Using direct cilia electrophysiology methods, we have determined that PKD2 expression is required for cilia channel function in the mouse collecting duct, but was not unaffected by PKD1 genetic repression. Thus, the impact of polycystin-1 on the cilia channel function is an open question in the human kidney. For this proposal we will utilize three human collecting duct cell lines –provided by the Mayo Clinic– which harbor nonsense germline variants W3263X, Q3978X and R4228X in PKD1. These three cell lines, which truncate the polycystin-1 protein prior to three potential polycystin-2 association sites (top, pore and coiled-coil domains), will be stably transfected with either the cilia fluorescent probe (ARL13B-RFP) or the cilia-specific calcium sensor (Smo-mCherry-GCaMP3). The resulting cell lines will be used to determine polycystin-1’s regulation of polycystin-2 cilia localization using super resolution imaging (Aim 1) and ion channel function using direct cilia electrophysiology and ratiometric calcium imaging (Aims 2 and 3). Since most germline ADPKD-causing variants (~85%) are found within the PKD1 gene, understanding their mechanistic impact on ciliary channels would define polycystin-1 function and provide new insights into their disease-causing mechanism. Potential mechanistic differences between these variants would support a rationale for personalized medicine for ADPKD and aid molecular diagnoses by helping to differentiate pathogenic mutations from neutral variants. Beyond ADPKD, variants in several cilia-localized proteins are associated with other forms of cystic kidney diseases. Thus, it is possible that multiple renal ciliopathies may share common aberrant cilia-to-cell signaling pathways, such as Ca2+ dysregulation. These results will firmly establish ADPKD not only as a ciliopathy but also as a channelopathy (a disease caused by an ion channel), where cystic pathology is initiated by aberrant Ca2+ signaling from the cilium.
Effective start/end date7/1/186/30/20


  • Mayo Clinic (NOR-217886/PO#66861701 // 5P30DK090728-09)
  • National Institute of Diabetes and Digestive and Kidney Diseases (NOR-217886/PO#66861701 // 5P30DK090728-09)


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