Molecular mechanism of the assembly of an acid-sensing receptor ion channel complex

Yu, Yong; Ulbrich, Maximilian H.; Li, Minghui; Dobbins, Scott; Zhang, Wei Kevin; Tong, Liang; Isacoff, Ehud Y.; Yang, Jian

doi:10.1038/ncomms2257

Cited by 49 publications

(84 citation statements)

References 51 publications

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“…It is generally believed that, in the polycystin complexes, TRPP subunits form an ion channel, whereas the PKD subunit receives the extracellular signal as a sensor or receptor (2,6). However, our recent study showed that PKD1L3 is also a channel pore-forming subunit (25), suggesting that other PKD proteins may also function as an ion channel subunit in their complexes with TRPPs.…”

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confidence: 78%

“…The downstream region of this trimer forms the PKD1-binding site and binds to one copy of the C-terminal coiled-coil domain of PKD1, determining a 3:1 (TRPP2/PKD1) subunit stoichiometry of the full-length complex (29,30). Our recent study concluded that this 3:1 stoichiometry is shared by the TRPP3-PKD1L3 complex (25), suggesting a common assembly mechanism among all polycystin complexes. Although several other domains and amino acids were found to be involved in TRPP2 homomeric assembly (31,32), so far, the C-terminal coiled-coil domain is the only site shown to be directly involved in its assembly with PKD1.…”

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confidence: 94%

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Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

Salehi-Najafabadi¹,

Li²,

Valentino

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellPolycystin complexes, or TRPP-PKD complexes, made of transient receptor potential channel polycystin (TRPP) and polycystic kidney disease (PKD) proteins, play key roles in coupling extracellular stimuli with intracellular Ca 2؉ signals. For example, the TRPP2-PKD1 complex has a crucial function in renal physiology, with mutations in either protein causing autosomal dominant polycystic kidney disease. In contrast, the TRPP3-PKD1L3 complex responds to low pH and was proposed to be a sour taste receptor candidate. It has been shown previously that the protein partners interact via association of the C-terminal or transmembrane segments, with consequences for the assembly, surface expression, and function of the polycystin complexes. However, the roles of extracellular components, especially the loops that connect the transmembrane segments, in the assembly and function of the polycystin complex are largely unknown. Here, with an immunoprecipitation method, we found that extracellular loops between the first and second transmembrane segments of TRPP2 and TRPP3 associate with the extracellular loops between the sixth and seventh transmembrane segments of PKD1 and PKD1L3, respectively. Immunofluorescence and electrophysiology data further confirm that the associations between these loops are essential for the trafficking and function of the complexes. Interestingly, most of the extracellular loops are also found to be involved in homomeric assembly. Furthermore, autosomal dominant polycystic kidney disease-associated TRPP2 mutant T448K significantly weakened TRPP2 homomeric assembly but had no obvious effect on TRPP2-PKD1 heteromeric assembly. Our results demonstrate a crucial role of these functionally underexplored extracellular loops in the assembly and function of the polycystin complexes.

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confidence: 78%

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confidence: 94%

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

Salehi-Najafabadi¹,

Li²,

Valentino

et al. 2017

Journal of Biological Chemistry

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“…TEVC experiments were performed as described previously (49). In brief, cRNAs were synthesized in vitro and injected into follicle membrane free oocytes (50 ng of cRNA per oocyte).…”

Section: Methodsmentioning

confidence: 99%

“…Western blot analysis was performed as described previously (49). Protein samples were run on 4-12% Bolt Bis-Tris Plus gels (Life Technologies) and transferred to a PVDF membrane.…”

Section: Methodsmentioning

confidence: 99%

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

Pavel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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

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Deciphering the Subunit Composition of Multimeric Proteins by Counting Photobleaching Steps

Arant

Ulbrich

2014

ChemPhysChem

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The limit of subdiffraction imaging with fluorescent proteins currently lies at 20 nm, and therefore most protein complexes are too small (2-5 nm) to spatially resolve their individual subunits by optical means. However, the number and stoichiometry of subunits within an immobilized protein complex can be resolved by the observation of photobleaching steps of individual fluorophores or co-localization of single-molecule fluorescence emission in multiple colors. We give an overview of the proteins that have been investigated by this approach and the different techniques that can be used to immobilize and label the proteins. This minireview should serve as a guideline for scientists who want to employ single-molecule subunit counting for their research.

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Molecular mechanism of the assembly of an acid-sensing receptor ion channel complex

Cited by 49 publications

References 51 publications

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

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

Deciphering the Subunit Composition of Multimeric Proteins by Counting Photobleaching Steps

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