Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain

Vien, Thuy N.; Wang, Jinliang; Ng, Leo C.T.; Cao, Erhu; DeCaen, Paul G.

doi:10.1073/pnas.1920777117

Cited by 31 publications

(29 citation statements)

References 87 publications

(114 reference statements)

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“…To test whether calcium occupancy of the EF hand is responsible for CDM and CDD of human polycystin-2, we generated double alanine mutations at the -X (T771A) and -Z vertices (E774A) which abolished Ca 2+ affinity for the CTD fragment ( Figure 1B). We then expressed the -X-Z double mutation in PKD2-GFP in a PKD2 null HEK cell line, so that exogenous channels could be assayed without the contribution of endogenous PKD2 alleles, as previously described (Vien et al, 2020). The stably expressed human WT and -X-Z channels trafficked to the primary cilia, and glass electrodes with submicron apertures were fabricated to form high resistance electrical seals with the GFP-illuminated cilia membranes (Figure 2A, Supplementary Movie 1).…”

Section: Resultsmentioning

confidence: 99%

Disrupting polycystin-2 EF hand Ca2+ affinity does not alter channel function or contribute to polycystic kidney disease

Vien

Smith

et al. 2020

Journal of Cell Science

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Approximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2. PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage dependent gating and subsequent desensitization— two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycytsin-2 intracellular EF hand is responsible for these forms of channel regulation, and if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest additional Ca2+ binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.

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Section: Resultsmentioning

confidence: 99%

Disrupting polycystin-2 EF hand Ca2+ affinity does not alter channel function or contribute to polycystic kidney disease

Vien

Smith

et al. 2020

Journal of Cell Science

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“…Although the results so far strongly suggest that the PC-1 CTL domain is critical for activating heterologous polycystin complexes, native polycystin complexes reside in the specialized membranes of primary cilia ( Liu et al, 2018 ; Kleene and Kleene, 2017 ; Vien et al, 2020 ). We, therefore, investigated whether the PC-1 N-terminus is able to activate polycystin complexes in the cilia of polarized mouse epithelial (mIMCD-3) cells using excised ciliary membrane patch clamp recordings ( Figure 5—figure supplement 1B ).…”

Section: Resultsmentioning

confidence: 98%

“…Next, we asked how the extracellular CTL domain in PC-1 might regulate activation of the entire channel complex. We reasoned that the TOP domain in PC-2 is well positioned to function as a surface receptor as it forms a bridge between the pore domain and voltage sensor-like domain, and is critical for channel activation ( Shen et al, 2016 ; Vien et al, 2020 ). In addition, different glycosylation patterns of the TOP domain might underlie conformational changes in the homomeric PC-2 complex ( Wilkes et al, 2017 ).…”

Section: Resultsmentioning

confidence: 99%

“…Both PC-1 and PC-2 have a large extracellular TOP domain, which contains prominent glycosylation sites. We speculate that the CTL domain in the native PC-1/PC-2 complex interacts with glycans in the TOP domain, which allosterically regulates the ion permeation pathway and/or gating apparatus of the heteromeric polycystin channel ( Vien et al, 2020 ; Figure 7 ). In particular, the N-glycan attached to N375 of PC-2 is adjacent to a structure that extends above TM3 and TM4 of the voltage sensor-like domain, so it is conceivable that binding of the CTL domain to this glycan will initiate conformational changes in the voltage sensor-like domain, which could be transmitted to the pore to regulate ion permeation.…”

Section: Discussionmentioning

confidence: 99%

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The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus

Nobuhara

Wang

et al. 2020

eLife

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Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here, we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.

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“…We showed that TgTRPPL-2 was able to conduct currents with conductance values comparable to the values of mammalian TRP channels [48–50]. Previous work with PC2, showed that Ca 2+ modulated the activity of PC2 [51–53] [48, 54].…”

Section: Discussionmentioning

confidence: 99%

Calcium signaling through a Transient Receptor Channel is important forToxoplasma gondiigrowth

Márquez-Nogueras

Chasen

Triana

et al. 2020

Preprint

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Transient Receptor Potential (TRP) channels participate in calcium (Ca2+) influx and intracellular Ca2+ release. TRP channels have not been studied in Toxoplasma gondii or any other Apicomplexan parasite. We characterized TgGT1_310560, a gene predicted to possess a TRP domain (TgTRPPL-2) and determined its role in Ca2+ signaling in T. gondii, the causative agent of toxoplasmosis. TgTRPPL-2 localized to the plasma membrane and the endoplasmic reticulum of T. gondii. ΔTgTRPPL-2 mutants were defective in growth and Ca2+ influx. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrated that TgTRPPL-2 is a non-selective cation channel that conducts Ca2+. Pharmacological blockers of TgTRPPL-2 inhibited Ca2+ influx and parasite growth. This is the first report of an Apicomplexan channel that conducts Ca2+ and initiates the Ca2+ signaling cascade that culminates in the stimulation of motility, invasion and egress. TgTRPPL-2 is a potential target for combating Toxoplasmosis.

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Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain

Cited by 31 publications

References 87 publications

Disrupting polycystin-2 EF hand Ca2+ affinity does not alter channel function or contribute to polycystic kidney disease

Disrupting polycystin-2 EF hand Ca2+ affinity does not alter channel function or contribute to polycystic kidney disease

The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus

Calcium signaling through a Transient Receptor Channel is important forToxoplasma gondiigrowth

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