. Autosomal dominant polycystic kidney disease (ADPKD) causes fatal progressive kidney failure and is commonly caused by variants in the PKD2 gene, which encodes the TRPP2 ion channel subunit1,2. Despite our strong understanding of the genetic basis of ADPKD, we still do not know how TRPP2 variants impact its ion channel function5,6. This basic question remains outstanding because TRPP2 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 results in real-time, and at super- and atomic resolution, thereby providing the most accurate description to date of ADPKD variants on TRPP2. We have published the first and highest resolution (3.0 ?) structure of TRPP2 using single-particle cryo-EM7, which has provided much-needed structural context for ADPKD-causing variants 8. TRPP2?s structure is unique among ion channels, having an extracellular lid-like component called the Top domain, which contains 59% of the germline variants found in the PKD2 gene. Within the Top domain is the Finger 1 motif, which is the site of 5 of the most pathogenic missense variants found in TRPP2 (R322Q/W, R325Q/P and C331S) and thus is the focus of our proposal8. We hypothesize that Finger 1 variants will cause loss of TRPP2 function due to one of two mechanisms: either defective TRPP2 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. We will validate these findings in situ using two animal models in Aim 2. Additionally, we will test the ?two-hit? model of ADPKD progression, which posits that this disease is recessive at the cellular level and cysts only develop after acquiring a second inactivating somatic mutation in the remaining allele3,4.
In Aim 3, we will concurrently assess how the variants alter TRPP2 channel assembly and the intramolecular interactions required for channel gating. The outcome of this project will determine the biophysical regulatory mechanism(s) of TRPP2 by the Top domain? an essential and defining component of this channel. By determining the impact of these variants at the molecular level, we will open the door to TRPP2- 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.
The proposal addresses the molecular and biophysical mechanism(s) controlling TRPP2 ion channel assembly and function at the ciliary membrane. We have developed novel tools to determine the structure and functional impact of ADPKD-causing variants found in the Finger 1 motif? results which have eluded researchers for the past 18 years. Our findings will determine if ADPKD is not only a ?ciliopathy? but also a ?channelopathy?, where cystic pathology is initiated by aberrant TRPP2 Ca2+ signaling from the cilium.
