Interaction between the S4–S5 linker and C-terminus of TRPP3 channel: functional importance and regulation by PIP2

Abstract

As one of the most common genetic renal diseases affecting over 12.5 million people worldwide, the autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either the PKD1 or PKD2 gene. Transient receptor potential polycystin-3 (TRPP3), a member of the polycystin protein family, also called polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), is a homologue of PKD2 but itself is not involved in ADPKD. A cation channel activated by calcium and protons, TRPP3 regulates calcium-dependent hedgehog signaling in primary cilia, intestinal development and sour tasting. It is an integral membrane protein featured with six transmembrane segments (S1-S6), a pore loop between S5 and S6, and intracellularly localized N- and C-termini. How TRPP3 channel function is regulated remains poorly understood. In this study, using the two-electrode voltage clamp (TEVC) electrophysiology in Xenopus laevis oocytes, Western blotting (WB), and immunofluorescence, I revealed three TRPP3 C-terminal domains critical for channel function: Ser581-Leu592, Arg596-Lys607 and Gly611-Thr622. We then identified conserved cationic residue (Lys 461) in the S4-S5 linker and aromatic residue (Tyr 564) in the C-terminal TRP-like domain of TRPP3 and studied their functional roles. While the physical proximity between the S4-S5 linker and TRP (-like) domain has been reported in many resolved TRP structures, it remains unknown whether there is physical and/or functional interaction. By co-immunoprecipitation (co-IP), in vitro pull-down, and blocking peptide strategy, we first revealed the physical and functional interaction between the S4-S5 linker and C-terminal TRP-like domain (L-C) of TRPP3 and then identified that it is mediated by a cationic-aromatic (K461-Y564) residue pair and seems to be inhibited by phosphatidylinositol 4,5- bisphosphate (PIP2). In summary, our studies constitute valuable contributions to the understanding of TRPP3 function and regulation.