ABSTRACT
28Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common systemic diseases and often leads to kidney failure and other life-threatening, extra-renal complications. Human genetic studies have firmly established that mutations in either PKD1 or PKD2, respectively encoding polycystin-1 and polycystin-2, cause most, if not all, ADPKD cases. An ample amount of knowledge regarding pathogenic mechanisms of ADPKD and physiological functions of polycystins has been obtained since the genes were positional cloned in the 1990s. More recently, structures of polycystin-2 and the polycystin-1/polycystin-2 complex have been determined, thanks to recent advancements in membrane protein biochemistry driven largely by application of mammalian cell-based expression systems and developments of artificial lipid bilayer systems and novel detergents and amphiphiles, as well as the “resolution revolution” in electron cryo-microscopy. In this chapter, we discuss the biochemical aspects of ADPKD and polycystins, structural features of polycystin-2 and the transient receptor potential ion channel family, as well as rapid evolution of electron cryo-microscopy as a predominant structure determination method. Step-by-step protocols of mammalian cell protein expression, purification, and structural determination are provided using polycystin-2 as an example.
