Direct measurement of polycystin channels and their regulation in cilia

Boston Children'S Hospital, Boston MA

Investigators

Abstract

? DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease is caused by mutations in the Pkd1 (85%) or Pkd2 (15%) genes. PKD morbidity is associated with primarily renal, but also liver, pancreatic, and vascular abnormalities such as intracranial aneurysms and aortic artery weakness. There are no effective therapies at present. Primary cilia house important regulators of cell growth and division. Defects in genes regulating cilia formation and function result in serious lifelong and adult-onset diseases. Here we focus on polycystins, the genes mutant in polycystic kidney diseases that are among the most common inherited disorders. The organizing principal of this proposal is that ciliary polycystin channels modify ciliary calcium levels that in turn modify ciliary signaling pathways regulating growth and cell division, and ultimately cyst formation. We measured ciliary calcium concentration and identified ion channel currents (Icilium) in single cilia. We found that calcium changes are largel confined to the cilium. We directly measured primary cilia currents under whole-cilium voltage clamp and found that the basal current was mediated by PC1-L1/PC2-L1 heteromers. These channels rapidly and dramatically alter cilia calcium levels. We were able to conduct these experiments by generating fluorescent indicators for calcium measurements that specifically target cilia. We showed that ciliary calcium levels regulate Smoothened-activated Gli1 transcription and ciliary tip accumulation of Gli2. We are crossing Pkd-mutant mice with these indicator mice. We focus on the function of the polycystin proteins themselves rather than downstream signal transduction pathways. Here we propose to measure ion channel currents in primary cilia from freshly isolated kidney collecting duct tubules, and tubular cells in intact tubules. The overall goal is to determine whether PC1 and PC2, the proteins mutant in polycystic kidney disease, also function as an ion channel complex in cilia. Our hypothesis is that PC-L1 proteins regulate cilia calcium to high levels and as cells mature and differentiate, they express PC proteins and reduce cilia calcium. In the experiments proposed, we will first identify the type of channel, PC or PC-L1, that is present in embryonic, neonatal, young adult, and mature adult mice. This requires careful evaluation of PC and PC-L1 biophysical and pharmacological properties. We will then determine and compare the domains of PC and PC-L1 that enable them to regulate calcium in cilia in distinct ways. Finally, we will determine how PC1, the more commonly affected protein in polycystic kidney disease, regulates the pore of the PC2-forming channel.