ABSTRACT
As a cation channel belonging to the transient receptor potential polycystin subfamily, polycystic kidney disease 2 protein (PKD2) functions as a homotetramer or a heterotetramer with PKD1. Pathogenic mutations in PKD2 or PKD1 account for the majority of the human autosomal dominant PKD cases, affecting 1 in 400–1000 individuals worldwide. Although PKD2 mutations or dysfunction have been extensively studied in terms of association with altered signaling pathways or disease manifestations over the past 20 years, how PKD2 functions as an ion channel has remained poorly understood, largely due to lack of a reliable function readout and known ligand. PKD2L1, which shares 54% amino acid identity with PKD2, is a nonselective cation channel involved in acid sensing. In this chapter, we describe the strategy and logic for identifying the hydrophobic activation gate of PKD2 and PKD2L1 channels through substitutions with hydrophilic residues to constitutively open the channels, by means of Xenopus laevis oocytes expression and the two-electrode voltage clamp technique. This allows obtaining a series of gain-of-function gate mutants of PKD2 and PKD2L1. We also discuss how ion permeation in the PKD channels is regulated by hydrophobic pore gates by comparing our functional studies with cryo-electron microscopy and molecular dynamics simulation analysis.
