. Autosomal dominant polycystic kidney disease (ADPKD) causes fatal progressive kidney failure and is commonly caused by variants in the PKD2 gene, which encodes the PKD2 ion channel subunit1,2. While considered a dominant monogenic disease, the prevailing ?two-hit hypothesis? of ADPKD progression states that this disease is recessive at the cellular level and cysts only develop after acquiring a second somatic mutation, which inactivates the remaining normal allele3,4. Despite our strong understanding of the genetic basis of ADPKD, we still do not know how PKD2 variants impact its ion channel function5,6. This basic question remains outstanding because PKD2 localizes to the enigmatic primary cilium?a tiny antenna-like organelle that requires innovative tools to study. To this end, we have developed novel tools to study cilia channels which yield quantifiable results, in real-time, and at super and atomic resolution, thereby providing the most accurate description to date of ADPKD variants on PKD2. We have published the first and highest resolution (3.0 ?) structure of PKD2 using single-particle cryo-EM7, which has provided much-needed structural context for ADPKD-causing variants in the PKD2 gene8. PKD2?s structure is unique among ion channels, having an extracellular lid-like component called the Top domain, which contains 59% of the germline missense variants found in the PKD2 gene. The Top domain contains the Finger 1 motif, which is the site of 5 of the most pathogenic missense variants found in PKD2 (R322Q/W, R325Q/P and C331S) and thus is the focus of our proposal8. We hypothesize that Finger 1 variants will cause loss of PKD2 function due to one of two mechanisms: either defective PKD2 channel gating (Hypothesis 1) or loss of ciliary trafficking due to impaired channel assembly (Hypothesis 2). In this proposal, we devise three specific aims to directly test these hypotheses. We will elucidate the impact of the Finger 1 variants on channel biophysics and ciliary localization using a heterologous method in Aim 1 and validate these findings in situ using two animal models in Aim 2. Concurrently we will interrogate the impact of Finger 1 variants on PKD2 channel assembly and the intramolecular interactions of the Top domain in Aim 3. Understanding the mechanistic and molecular implications of these variants will open the door to PKD2- targeted rational drug design for the treatment of ADPKD9. 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 diseases10,11. Thus, it is possible that multiple renal ciliopathies may share common aberrant cilia-to-cell signaling pathways, such as Ca2+ dysregulation12,13. 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 cilium14-17.
This proposal addresses the fundamental effect of ADPKD-causing variants on ciliary PKD2 ion channels, which has eluded researchers for the past 18 years. We have developed novel tools to study heterologous and native cilia channels ?which yield quantifiable results, in real-time, and at super resolution? thereby providing the most accurate description of ADPKD variants in PKD2. 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 controlled by aberrant Ca2+ signaling from the cilium.
