This proposal will support the candidate's career goals of studying PKD (polycystic kidney disease) calcium channels proposed to be present in the primary cilia of kidney collecting duct cells and their inactivation by internal calcium (Ca2+) The candidate has developed and published two innovative techniques to measure ion channel events in the primary cilium, which distinguishes his research from others in the field. First, the candidate developed electrophysiology methods that measure the membrane potential of the cilia compartment and ionic flux across the membrane. Second, the candidate applied a genetically encoded ratiometric Ca-sensor to detect calcium changes within the cilium. The candidate has demonstrated the effectiveness of these methods, identifying the PKD1-L1/2-L1 ion channel present in the primary cilium of non- renal cells. For the duration of the K99/R00 grant, the candidate proposes to apply these methods to the primary cilia of kidney cells to determine the genetic identify the ion channels present. Although the candidate is training in a laboratory with FRET experience, the candidate will take on additional training in FRET experimentation and analysis to be applied in the following aim. The candidate will use the FRET methods in conjunction with electrophysiology to dissect the molecular mechanisms by which Ca2+ inactivates PKD channels. The candidate will use this project to lay the groundwork for his long-term goal of identifying genes, such as Pkd, which result in the development of polycystic kidney diseases. During the latter phase of the K99 period the candidate will train in genomic and bioinformatic methods necessary for isolating heritable genetic factors that may lead to the development of polycystic disease. In conjunction with the tools he has developed and additional training, the candidate will have a wide range of methods to identify and determine the function of potential genes which may alter the ion channel within the primary cilium. The candidate is well-qualified to carry out the short- and long-term goals described above. He has over ten years of experience in ion channel biology, has spent considerable effort learning and developing methods to study the primary cilium. If awarded, the candidate plans on finding a tenured faculty position in an environment supported by experts in cellular physiology and genetic approaches.
Mutations in Polycystic Kidney Disease proteins 1 and 2 (PKD1 and PKD2) can cause kidney cysts and renal failure. PKD1 and PKD2 are proposed interact and form ion channels in the primary cilia of the collecting duct cells of the kidney. However the activity of PKD1/ 2 channels in the cilium has not been measured. Thus their function on the primary cilium has not been determined. In this proposal I will determine the molecular identity of the ion channel present on primary cilia of the kidney collecting duct cells and how they are regulated by internal calcium.
| Naylor, Claire E; Bagnéris, Claire; DeCaen, Paul G et al. (2016) Molecular basis of ion permeability in a voltage-gated sodium channel. EMBO J 35:820-30 |
