The genetic identity of the primary cilia ion channels of kidney collecting duct cells and their regulation by internal calcium

This summary describes the candidate’s progress during the K99 mentored phase and proposed transition to R00 phase of the grant. The candidate’s goals are to study PKD (polycystic kidney disease) ion channels proposed to be present in the primary cilia of kidney collecting duct cells and modulation by calcium (Ca2+). The candidate has deployed two innovative techniques to measure ion channel events in the primary cilium: genetically coded-radiometric Ca2+ sensors and cilia voltage clamp. During the mentored phase, the candidate has genetically identified PKD2 as subunit of the current found in cilia of inner medullary collecting duct epithelial (IMCD) cells of the kidney. We have generated significant preliminary data that defines the type of ionic which pass through the channel as well as it’s biophysical properties. This year, we published a novel mechanism by which Ca2+ occupies the selectivity filter of PKD2-l1 which will likely extend to the PKD2 channel as well. The candidate has overcome the lack of heterologous membrane expression of PKD2 by generating chimeras and recording the overexpressed channel in cilia heterologously. We have made significate progress determining the structure of the PKD2 channel (unpublished), which will provide a structural basis to understand disease-causing mutations in Pkd2. For the duration of the R00 phase, the candidate proposes to apply these tools to screen for drug compounds which modulate PKD2 function thereby potentially identifying therapeutically useful compounds for the treatment of ADPKD. The candidate has trained and developed FRET methods to determine calmodulins association with PKD2 effects channel states to be deployed in the R00 phase. The candidate has trained and will continue to train in genomic and bioinformatic methods necessary for isolating heritable genetic factors that may lead to the development of polycystic disease. The candidate will use these tools in the R00 period for his long-term goal of identifying genes, such as Pkd, which result in the development of polycystic kidney diseases. The candidate has over eleven years of experience in ion channel biology, has spent considerable effort learning and developing methods to study the primary cilia during his independent phase of the grant.