Functional roles of glutamic acid E143 and E705 residues in the N-terminus and transmembrane domain 7 of Anoctamin 1 in calcium and noxious heat sensing

Choi, Jonghyun; Jang, Yongwoo; Kim, Hae-Dong; Wee, Jungwon; Cho, Sinyoung; Son, Woo Sung; Kim, Sung Min; Yang, Young Soon

doi:10.5483/bmbrep.2018.51.5.199

Cited by 4 publications

(3 citation statements)

References 30 publications

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“…For example, YFP-H148Q/I152L has been used as a sensor for I – concentration changes produced by the sodium–iodide symporter (NIS) . Additionally, separate studies measured I – influx through glycine and GABA A receptor chloride channels using YFP-I152L and YFP-V163S and the anoctamin 1 (ANO1) anion channel using YFP-F46L/H148Q/I152L . Our results add to the reports that underline the usefulness of YFP-based anion sensors for the investigation of anion channels.…”

Section: Discussionsupporting

confidence: 61%

Genetically Encoded Halide Sensor-Based Fluorescent Assay for Rapid Screening of Glutamate Transport and Inhibition

Zielewicz

Grewer

2019

ACS Sens.

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Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system. Excitatory amino acid transporters (EAATs) are a family of transmembrane transporters responsible for glutamate uptake into cells, and their malfunction is related to a variety of diseases, including neurodegenerative diseases and stroke. Screening for and developing inhibitors of EAATs as well as related transporters is a significant field of study for biomedical and pharmaceutical applications. Rapid, highthroughput methods are critical for the study of glutamate transporters, and fluorescent methods are appealing for this purpose as compared to more traditional electrophysiological methods. In this study, we present a method for studying glutamate transporters and inhibitors by utilizing a mutated version of a yellow fluorescent protein (YFP) highly sensitive to quenching by anions (mClY). We applied this YFP variant to fluorescent imaging of anion flux in HEK293 cells caused by transiently expressed excitatory amino acid carrier 1 (EAAC1) and excitatory amino acid transporter 2 (EAAT2) and its inhibition by competitive blockers. This method enables rapid identification of inhibitors and, potentially, activators of EAAT function, which is critical for glutamate transport research.

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Section: Discussionsupporting

confidence: 61%

Genetically Encoded Halide Sensor-Based Fluorescent Assay for Rapid Screening of Glutamate Transport and Inhibition

Zielewicz

Grewer

2019

ACS Sens.

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“…Next, we examined the protein-protein interaction between TTYH2 and β-COP at the single-cell level using the bimolecular fluorescence complementation (BiFC) assay. The BiFC assay is a useful technique to visualize the protein-protein interaction and its subcellular localization in a live cell (8, 9). We constructed three expressing vectors (TTYH2-VN, TTYH2ΔC-VN, and VC-β-COP), which were the N- or C-terminal tagged with N-terminal halve (VN) or C-terminal halve (VC) of the split Venus fluorescent protein.…”

Section: Resultsmentioning

confidence: 99%

Surface expression of TTYH2 is attenuated by direct interaction with β-COP

et al. 2019

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TTYH2 is a calcium-activated, inwardly rectifying anion channel that has been shown to be related to renal cancer and colon cancer. Based on the topological prediction, TTYH2 protein has five transmembrane domains with the extracellular N-terminus and the cytoplasmic C-terminus. In the present study, we identified a vesicle transport protein, β-COP, as a novel specific binding partner of TTYH2 by yeast two-hybrid screening using a human brain cDNA library with the C-terminal region of TTYH2 (TTYH2-C) as a bait. Using in vitro and in vivo binding assays, we confirmed the protein-protein interactions between TTYH2 and β-COP. We also found that the surface expression and activity of TTYH2 were decreased by co-expression with β-COP in the heterologous expression system. In addition, β-COP associated with TTYH2 in a native condition at a human colon cancer cell line, LoVo cells. The over-expression of β-COP in the LoVo cells led to a dramatic decrease in the surface expression and activity of endogenous TTYH2. Collectively, these data suggested that β-COP plays a critical role in the trafficking of the TTYH2 channel to the plasma membrane.

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“…As we have previously shown, ANO1 channel activity was measured by the halide-sensitive YFP imaging technique. This was done by using a cell line that stably expresses a genetically encoded iodide-sensing fluorescent protein YFP (F46L/H148Q/I152L) and ANO1 [34]. Changes in the concentration of cytoplasmic iodide alter the fluorescence intensity of the iodide-sensitive YFP; extracellular iodide influx induced by ANO1 activation leads to a decrease in fluorescence intensity.…”

Section: Identification Of a Novel Aa3 As Ano1 Inhibitormentioning

confidence: 99%

Design of Anticancer 2,4-Diaminopyrimidines as Novel Anoctamin 1 (ANO1) Ion Channel Blockers

Kim

Cho

et al. 2020

Molecules

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Pyrimidine is a privileged scaffold in many synthetic compounds exhibiting diverse pharmacological activities, and is used for therapeutic applications in a broad spectrum of human diseases. In this study, we prepared a small set of pyrimidine libraries based on the structure of two hit compounds that were identified through the screening of an in-house library in order to identify an inhibitor of anoctamin 1 (ANO1). ANO1 is amplified in various types of human malignant tumors, such as head and neck, parathyroid, and gastrointestinal stromal tumors, as well as in breast, lung, and prostate cancers. After initial screening and further structure optimization, we identified Aa3 as a dose-dependent ANO1 blocker. This compound exhibited more potent anti-cancer activity in the NCI-H460 cell line, expressing high levels of ANO1 compared with that in A549 cells that express low levels of ANO1. Our results open a new direction for the development of small-molecule ANO1 blockers composed of a pyrimidine scaffold and a nitrogen-containing heterocyclic moiety, with drug-like properties.

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Functional roles of glutamic acid E143 and E705 residues in the N-terminus and transmembrane domain 7 of Anoctamin 1 in calcium and noxious heat sensing

Cited by 4 publications

References 30 publications

Genetically Encoded Halide Sensor-Based Fluorescent Assay for Rapid Screening of Glutamate Transport and Inhibition

Genetically Encoded Halide Sensor-Based Fluorescent Assay for Rapid Screening of Glutamate Transport and Inhibition

Surface expression of TTYH2 is attenuated by direct interaction with β-COP

Design of Anticancer 2,4-Diaminopyrimidines as Novel Anoctamin 1 (ANO1) Ion Channel Blockers

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