Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant

Abstract

Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/ polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca 2+, inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing singlepoint ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.autosomal dominant polycystic kidney disease | TRP channels | TRPP2 | polycystin | gain of function

The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs

“…Electrophysiology studies have suggested that heterologous PKD2 currents can be measured from the plasma membrane when it is either expressed with PKD1 in mammalian cells or when mutated (F604P) to render it constitutively active in Xenopus laevis oocytes (Arif Pavel et al, 2016; Hanaoka et al, 2000). However, we find that currents from transfected HEK293T and CHO cells were not different from untransfected cells when measured under physiological calcium or divalent-free sodium conditions (Figures S4A and S4B).…”

“…Percent change of the outward current was determined by the following equation: % Change I out = (I drug -I control /I control ) × 100 where I control is the average current measured during the 30 s prior to drug application and I drug is the average current after 60–120 s of drug application. The human PKD2 F604P mutant (Arif Pavel et al, 2016) was recorded in DVF symmetrical 140 mM Na + conditions. Non-transfected HEK293T cells exhibit small outwardly rectifying currents in physiological calcium conditions, which become larger and ohmic (linear) in divalent free conditions.…”

“…The PKD2 ion channel initially was reported to conduct calcium (Hanaoka et al, 2000) and later shown to be blocked by calcium (Cai et al, 2004). Most recently, a gain-of-function mutation, but not the wild-type PKD2, underlies a measurable current when heterologously expressed in Xenopus oocytes (Arif Pavel et al, 2016). Another study claims that it is indirectly activated by Wnt ligands via PKD1 (Kim et al., 2016).…”

Progress in ciliary ion channel physiology

“…These results contrast with reports that heterologous PKD2 (PKD1 + PKD2) is selective for calcium. The proposed location of F604 is in the fifth transmembrane helix of PKD2, and thus, the activating F604P mutation may create a bend in the activation gate, opening the pore and activating the channel (Arif Pavel et al, 2016). Given the lack of function of WT PKD2, these data suggest a significant conformational change must occur for the channel to open as supported by recent structural studies (Shen et al, 2016).…”

Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2 (PC2)

“…Most other cases of ADPKD (70%) are caused by mutations in PC1, which forms a 1:3 complex with PC2 by replacing one PC2 subunit in the channel region (Su et al, 2018a). Phenotypically, ADPKD is characterized by formation of fluid-filled renal cysts, which leads to progressive cystic enlargement in both kidneys and ultimately kidney failure (Pavel et al, 2016). Cysts or diverticula also frequently develop in intestines, liver and pancreas (Wilson, 2004).…”